U.S. patent application number 13/946959 was filed with the patent office on 2014-01-23 for compounds, compositions, and methods for cancer therapy.
The applicant listed for this patent is The Broad Institute, Inc.. Invention is credited to Drew Adams, Zarko Boskovic, Mingji Dai, Mahmud Mustaqim Hussain, Stuart Schreiber.
Application Number | 20140024639 13/946959 |
Document ID | / |
Family ID | 49947057 |
Filed Date | 2014-01-23 |
United States Patent
Application |
20140024639 |
Kind Code |
A1 |
Adams; Drew ; et
al. |
January 23, 2014 |
Compounds, Compositions, and Methods for Cancer Therapy
Abstract
Compounds including various oligomers of piperlongumine and/or
piperlongumine analogues as well as certain piperlongumine
analogues that exhibit improved toxicity to cancer cells are
disclosed. Also provided are compositions that comprise the
compounds, methods of making compositions comprising the compounds,
methods of making the compounds, and the use of compounds in
methods for treating cancer.
Inventors: |
Adams; Drew; (Cambridge,
MA) ; Dai; Mingji; (West Lafayette, IN) ;
Schreiber; Stuart; (Boston, MA) ; Hussain; Mahmud
Mustaqim; (Cambridge, MA) ; Boskovic; Zarko;
(Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Broad Institute, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
49947057 |
Appl. No.: |
13/946959 |
Filed: |
July 19, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61779434 |
Mar 13, 2013 |
|
|
|
61674100 |
Jul 20, 2012 |
|
|
|
Current U.S.
Class: |
514/212.03 ;
514/328; 514/336; 514/337; 514/340; 514/350; 514/423; 540/529;
546/220; 546/257; 546/280.4; 546/281.1; 546/283.1; 546/298;
564/159 |
Current CPC
Class: |
C07D 207/38 20130101;
A61K 31/4412 20130101; C07D 409/04 20130101; C07D 405/06 20130101;
C07D 211/74 20130101; C07D 401/04 20130101; C07C 233/91 20130101;
C07D 225/02 20130101; A61K 31/4015 20130101; C07D 223/10 20130101;
C07D 409/06 20130101; A61K 31/4015 20130101; A61K 31/4436 20130101;
A61K 31/45 20130101; A61K 31/55 20130101; A61K 31/4436 20130101;
A61K 45/06 20130101; A61K 31/55 20130101; C07D 211/86 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/444
20130101; A61K 31/444 20130101; A61K 31/4412 20130101 |
Class at
Publication: |
514/212.03 ;
546/298; 540/529; 546/220; 546/281.1; 546/280.4; 546/257;
546/283.1; 564/159; 514/350; 514/328; 514/336; 514/340; 514/337;
514/423 |
International
Class: |
C07D 211/86 20060101
C07D211/86; C07D 211/74 20060101 C07D211/74; C07D 409/06 20060101
C07D409/06; C07D 409/04 20060101 C07D409/04; C07D 401/04 20060101
C07D401/04; A61K 45/06 20060101 A61K045/06; C07C 233/91 20060101
C07C233/91; A61K 31/4412 20060101 A61K031/4412; A61K 31/55 20060101
A61K031/55; A61K 31/45 20060101 A61K031/45; A61K 31/4015 20060101
A61K031/4015; C07D 225/02 20060101 C07D225/02; C07D 405/06 20060101
C07D405/06 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
R01GM038627, awarded by the National Institutes of Health. The
government has certain rights to this invention.
Claims
1. A compound having the formula: ##STR00227## wherein A is C(O) or
S(O).sub.2; wherein B is an alkyl, alkenyl, or alkynyl; wherein
n=0, 1, 2, or 3; wherein R.sub.1 is selected from the group
consisting of a halogen, hydrogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl, a
C.ident.C-aryl halide, and an aryl group; wherein R.sub.2 is
selected from the group consisting of hydrogen, alkyl, alkenyl, and
an aryl group; wherein R.sub.3 is selected from the group
consisting of hydrogen, alkyl, alkenyl, and an aryl group; and,
wherein R.sub.4 is selected from the group consisting of hydrogen,
alkyl, a compound of the formula: ##STR00228## wherein B is alkenyl
or alkynyl, R.sub.5 is selected from the group consisting of
hydrogen, halogen, and methoxy, and wherein each of R.sub.6,
R.sub.7, and R.sub.8 are independently selected from the group
consisting of hydrogen, bromine, chlorine, fluorine, keto,
hydroxyl, alkyl, alkenyl, alkoxy, an aminoalkenyl, and an
aminoalkoxy group with the proviso that at least one of R.sub.5,
R.sub.6, and R.sub.7 is not a methoxy group when A is C(O), n=0,
and R.sub.1, R.sub.2, and R.sub.3 are hydrogen, and with the
proviso that at least one of R.sub.6, R.sub.7, and R.sub.8 is not a
hydrogen, methoxy group, alkoxy, or aminoalkoxy group when R.sub.1,
R.sub.2, and R.sub.3 are hydrogen, and a compound of the formula:
##STR00229## wherein A is C(O) or S(O).sub.2, and is optionally
joined to B by X; wherein X, when present, is an alkyl, alkenyl,
alkynyl, aryl, or combination thereof; wherein n=0, 1, 2, or 3;
wherein R.sub.9 is selected from the group consisting of a halogen,
hydrogen, C.ident.C-alkyl, C.ident.C-cycloalkyl,
C.ident.C-cycloakyl halide, C.ident.C-aryl, a C.ident.C-aryl
halide, and an aryl group; wherein R.sub.10 is selected from the
group consisting of hydrogen, alkyl, alkenyl, and an aryl group;
and, wherein R.sub.11 is selected from the group consisting of
hydrogen, alkyl, alkenyl, and an aryl group.
2. The compound of claim 1, wherein R4 is independently selected
from the group consisting of hydrogen, alkyl, phenyl, and a
compound of the formula: ##STR00230## wherein A is C(O) or
S(O).sub.2, and is optionally joined to B by X, wherein X, when
present, is an alkyl, alkenyl, alkynyl, aryl, or combination
thereof; wherein n=0, 1, 2, or 3; wherein R.sub.9 is selected from
the group consisting of a halogen, hydrogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl, a
C.ident.C-aryl halide, and an aryl group; wherein R.sub.10 is
selected from the group consisting of hydrogen, alkyl, alkenyl, and
an aryl group; and, wherein R.sub.11 is selected from the group
consisting of hydrogen, alkyl, alkenyl, and an aryl group.
3. The compound of claim 1, wherein R.sub.1 is a
C.ident.C-cycloalkyl group wherein said cycloalkyl is a C3 to C6
ring and/or wherein R.sub.3 is hydrogen or a thienyl group.
4. The compound of claim 3, wherein R.sub.1 is a
C.ident.C-cycloalkyl, wherein said cycloakyl is substituted at the
ring carbon that is bound to the alkynyl carbon with a hydroxyl
group and/or wherein R.sub.3 is hydrogen or a thienyl group.
5. The compound of claim 1, wherein R.sub.1 is a C.ident.C-phenyl
or a C.ident.C-phenyl halide wherein the halide is substituted
ortho or para to the phenyl ring carbon that is bound to the
alkynyl carbon and/or wherein R.sub.3 is hydrogen or a thienyl
group.
6. The compound of claim 2, wherein R.sub.1 and R.sub.9 are
independently selected from a group consisting of hydrogen, a
halogen, and a C.ident.C-cycloalkyl group wherein said cycloalkyl
is a C3 to C6 ring and/or wherein R.sub.3 and R11 are independently
selected from a group consisting of hydrogen and a thienyl
group.
7. The compound of claim 6, wherein R.sub.1 and R.sub.9 are
independently selected from the group consisting of hydrogen, a
halogen, and a C.ident.C-cycloalkyl group, wherein said cycloakyl
is substituted at the ring carbon that is bound to the alkynyl
carbon with a hydroxyl group and/or wherein R.sub.3 is hydrogen or
a thienyl group.
8. The compound of claim 2, wherein R.sub.1 and R.sub.9 are
independently selected from the group consisting of hydrogen, a
halogen, and is a C.ident.C-phenyl or a C.ident.C-phenyl halide
wherein the halide is substituted ortho or para to the phenyl ring
carbon that is bound to the alkynyl carbon and/or wherein R.sub.3
is hydrogen or a thienyl group.
9. The compound of claim 1, wherein said compound has a formula
selected from the group consisting of: ##STR00231## ##STR00232##
##STR00233##
10. A compound of the formula: ##STR00234## wherein R.sub.1 is
either: (i) halogen, C.ident.C-alkyl, C.ident.C-cycloakyl,
C.ident.C-cycloakyl halide, C.ident.C-aryl, a C.ident.C-aryl
halide, or an aryl group; or, (ii) an alkyl, thioalkyl, aminoalkyl,
or alkenyl group that is optionally fused to R.sub.2 when R.sub.2
is alkyl and wherein the ring formed by the fusion consists of 5,
6, 7, or 8 members; wherein R.sub.2 is alkyl; wherein R.sub.3 is
alkyl; wherein R.sub.4 is selected from the group consisting of
hydrogen, halogen, C.ident.C-alkyl, C.ident.C-cycloalkyl,
C.ident.C-cycloakyl halide, C.ident.C-aryl, a C.ident.C-aryl
halide, and an aryl group; and, wherein R.sub.5 is selected from
the group consisting of hydrogen, alkyl, S(O).sub.2--R.sub.6, and
C(O)--R.sub.6, wherein R.sub.6 is alkyl, alkenyl, or alkynyl.
11. The compound of claim 10, wherein said compound has the
structure: ##STR00235##
12. A composition comprising the compound of claim 1 and a
pharmaceutically acceptable excipient.
13. The composition of claim 12, wherein said composition further
comprises an additional chemotherapeutic agent.
14. A method of treating cancer comprising administration of a
therapeutically effective amount of the compound of claim 1 to a
subject in need thereof.
15. The method of claim 14, where said method further comprises
administration of an additional chemotherapeutic agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This U.S. Non-Provisional patent application claims the
benefit of U.S. Provisional Patent Application No. 61/779,434,
filed Mar. 13, 2013, which is incorporated herein by reference in
its entirety, and the benefit of U.S. Provisional Patent
Application No. 61/674,100, filed Jul. 20, 2012, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0003] Reactive oxygen species (ROS) are natural byproducts of
oxidative respiration and can function in signal transduction and
clearance of pathogens during innate immune responses. Cancer cells
have been reported to harbor higher levels of ROS than
non-transformed cells, and in some cases activation of a specific
oncogene (for example, HRAS) is sufficient to elevate levels of ROS
(1, 2). Since ROS are capable of damaging crucial cellular
macromolecules, including DNA, some cancer cells may be faced with
chronic `oxidative stress` that requires active enzymatic ROS
detoxification to prevent induction of cell death. As such, one
consequence of some of the genomic alterations leading to
tumorigenesis may be a dependency on pathways facilitating the
detoxification of ROS for survival, a form of `nononcogene
addiction` or `non-oncogene co-dependency` (3-5). Importantly, this
dependency might not be shared by many non-transformed cells, whose
lower basal ROS levels and/or elevated antioxidant capacity could
provide resistance to treatments that impair ROS metabolism.
Various small molecules, including many with disulfide,
-unsaturated carbonyl, sulfonate, or other electrophilic functional
groups, have previously been shown to elevate ROS levels and induce
cancer cell death (6). A subset of such compounds has also
demonstrated a degree of selective toxicity toward cancer cells in
in vitro and in vivo models (7-12).
[0004] A cell-based, high-throughput screening approach was used to
identify piperlongumine (PL), a naturally occurring, electrophilic
small molecule capable of selectively killing a variety of
transformed cell types while sparing primary normal cells (5).
Piperlongumine's in vivo antitumor efficacy was illustrated in
mouse models of cancer, including xenograft and spontaneous mammary
tumor formation models. Mechanistic investigations correlated the
observed selective toxicity with a cancer-selective increase in ROS
and other markers of oxidative stress following treatment with PL,
as well as increases in DNA damage and apoptotic cell death (FIG.
1A). The small-molecule nucleophile and antioxidant
N-acetyl-L-cysteine prevents PL-mediated cell death, and several
proteins known to bind glutathione and detoxify ROS were identified
as potential cellular interaction partners of PL through affinity
purification and quantitative proteomics.
[0005] Piperlongumine analogs where methoxy groups of
piperlongumine are substituted with hydrogen, hydroxy, methyl, or
other groups have been described (UA 2009/0312373; UA 2011/0053938;
UA 2012/0059004, UA 2012/0157455; Duh et al. J. Nat. Prod. 1990
November-December, 53(6) 1575-1577; Duh et al., Phytochemistry
1990, 29: 2689-2691; Raj et al. Nature 475, 231-234 (2011)).
SUMMARY
[0006] Compounds comprising oligomers of piperlongumine (PL) and/or
piperlongumine analogues (PLA) as well as certain PLA compounds
that exhibit improved toxicity to cancer cells are provided herein.
The compounds provided herein are collectively referred to herein
as "iPLA" compounds (for "improved piperlongumine analogues"). In
certain embodiments, such improvements exhibited by iPLA compounds
can comprise about a 10- to 2-fold decrease in EC.sub.50 values for
cancer cell toxicity relative to the corresponding EC.sub.50 values
for PL. In certain embodiments, such improvements in EC.sub.50
values for cancer cell toxicity exhibited by iPLA compounds are
accompanied by selective toxicity towards cancer cells that is
comparable to that exhibited by PL. In certain embodiments, iPLA
compounds are accompanied by selective toxicity towards cancer
cells that is improved relative to that exhibited by PLA are
provided. Also provided herein are compositions comprising iPLA
compounds, methods of making compositions comprising the iPLA
compounds, methods of making iPLA compounds, and the use of iPLA
compounds in methods for treating cancer.
[0007] Oligomers comprising monomers independently selected from
the group consisting of a piperlongumine (PL) monomer, a
piperlongumine analog (PLA) monomer, and pharmaceutically
acceptable salts thereof, wherein said monomers are covalently
linked at a position in their respective carbon chains that is
independently selected from the group consisting of the C11
position, the C12 position, and the C13 position are provided. In
certain embodiments, the monomers are linked via a chain selected
from the group consisting of alkyl, alkenyl, alkoxy, aminoalkyl,
aminoalkenyl, and an aminoalkoxy group. In certain embodiments, the
monomers are linked via one or more chain(s) independently selected
from the group consisting of alkyl, alkenyl, alkoxy, aminoalkyl,
aminoalkenyl, and an aminoalkoxy group, wherein at least one of
said groups are joined in said chain via an ether, ester,
thioester, thioether, amide, or keto group. In certain embodiments,
the C11, C12, or C13 positions are linked by a 3 to 15 atom chain.
In certain embodiments, the chain is a branched chain. In certain
embodiments, the aminoalkoxy group is of the formula
--O--(CH.sub.2).sub.n1--N(R.sub.7)--(CH.sub.2).sub.n2--O--, wherein
n.sub.1=1-6, n.sub.2=1-6, and R.sub.7 is selected from the group
consisting of hydrogen, alkyl, alkenyl, alkoxy, aminoalkyl,
aminoalkenyl, aminoalkoxy, and an aminoalkoxy group. In certain
embodiments, a first and a second PL or PLA monomer are linked by
an aminoalkoxy group is of the formula
--O--(CH.sub.2).sub.n1--N(R.sub.7)--(CH.sub.2).sub.n2--O--, wherein
n.sub.1=1-6, n.sub.2=1-6, and R.sub.7 is selected from the group
consisting of alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl,
aminoalkoxy, wherein said R.sub.7 group is linked to a third PL
monomer or to a third PLA monomer. In certain embodiments, the,
oligomer is a dimer or a trimer. In certain embodiments, the PLA
monomer is a compound having the formula:
##STR00001##
wherein A is C(O) or S(O).sub.2; wherein n=0, 1, 2, or 3; wherein
the ortho-carbon of the phenyl ring is unsubstituted or substituted
with a halogen; wherein R.sub.1 is selected from the group
consisting of hydrogen, halogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl,
C.ident.C-aryl halide, and an aryl group; wherein R.sub.2 is
selected from the group consisting of hydrogen, alkyl, alkenyl, and
an aryl group; wherein R.sub.3 is selected from the group
consisting of hydrogen, alkyl, alkenyl, and an aryl group; and,
wherein each of R.sub.4, R.sub.5, and R.sub.6 is independently
selected from the group consisting of hydrogen, bromine, chlorine,
fluorine, keto, hydroxyl, alkyl, alkenyl, alkoxy, aminoalkyl,
aminoalkenyl, and an aminoalkoxy group, with the proviso that said
compound is not a PL monomer. In certain embodiments, R.sub.1 is a
C.ident.C-cycloalkyl group, wherein said cycloalkyl is a C3 to C6
ring. In certain embodiments, R.sub.1 is a C.ident.C-cycloalkyl,
wherein said cycloakyl is substituted at the ring carbon that is
bound to the alkynyl carbon with a hydroxyl group. In certain
embodiments, R.sub.1 is a C.ident.C-phenyl or a C.ident.C-phenyl
halide wherein a halide is substituted ortho or para to the phenyl
ring carbon that is bound to the alkynyl carbon. In certain
embodiments, the oligomer is a compound of a formula selected from
the group consisting of:
##STR00002##
wherein R is selected from the group consisting of C1-C4 alkyl,
--C(O)--(CH.sub.2)n-COOH where n=1-4, and salts thereof.
[0008] Also provided are compositions comprising any of the
aforementioned oligomers and a pharmaceutically acceptable
excipient. In certain embodiments, the compositions can further
comprise an additional chemotherapeutic agent.
[0009] Also provided are methods of treating cancer comprising
administration of a therapeutically effective amount of any of the
aforementioned oligomers to a subject in need thereof. In certain
embodiments, the methods can further comprise administration of an
additional chemotherapeutic agent.
[0010] Also provided are methods of making an oligomer of a
piperlongumine (PL) monomer and/or a piperlongumine analog (PLA)
monomer, comprising the steps of reacting an alkyl diol, an
aminoalkyl diol, an alkyl triol, or an aminoalkyl triol with
piperlongumine or piperlongumine analogue under conditions that
provide for ether bond formation between said hydroxyl group and
said diols or triols, wherein at least one of the C11, C12, or C13
positions of said piperlongumine (PL) monomer and/or a
piperlongumine analog (PLA) monomer is substituted with a hydroxyl
group.
[0011] Also provided are compounds having the formula:
##STR00003##
wherein A is C(O) or S(O).sub.2; wherein n=0, 1, 2, or 3; wherein
the ortho-carbon of the phenyl ring is unsubstituted or substituted
with a halogen; wherein R.sub.1 is selected from the group
consisting of a halogen, C.ident.C-alkyl, C.ident.C-cycloalkyl,
C.ident.C-cycloakyl halide, C.ident.C-aryl, a C.ident.C-aryl
halide, and an aryl group; wherein R.sub.2 is selected from the
group consisting of hydrogen, alkyl, alkenyl, and an aryl group;
wherein R.sub.3 is selected from the group consisting of hydrogen,
alkyl, alkenyl, and an aryl group; and, wherein each of R.sub.4,
R.sub.5, and R.sub.6 is independently selected from the group
consisting of hydrogen, bromine, chlorine, fluorine, keto,
hydroxyl, alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl, and an
aminoalkoxy group. In certain embodiments, R.sub.1 is a
C.ident.C-cycloalkyl group wherein said cycloalkyl is a C3 to C6
ring. In certain embodiments, R.sub.1 is a C.ident.C-cycloalkyl,
wherein said cycloakyl is substituted at the ring carbon that is
bound to the alkynyl carbon with a hydroxyl group. In certain
embodiments, compound of claim 19, R.sub.1 is a C.ident.C-phenyl or
a C.ident.C-phenyl halide wherein a halide is substituted ortho or
para to the phenyl ring carbon that is bound to the alkynyl carbon.
In certain embodiments, the compound has a formula selected from
the group consisting of:
##STR00004## ##STR00005## ##STR00006##
[0012] Also provided are compositions comprising any of the
aforementioned compounds having the formula:
##STR00007##
and a pharmaceutically acceptable excipient. In certain
embodiments, the composition can further comprise an additional
chemotherapeutic agent.
[0013] Also provided are methods of treating cancer comprising
administration of a therapeutically effective amount of any of the
aforementioned compounds having the formula:
##STR00008##
to a subject in need thereof. In certain embodiments, the methods
can further comprise administration of an additional
chemotherapeutic agent.
[0014] Also provided is a compound with the formula:
##STR00009##
[0015] Also provided are compositions comprising the compound with
the formula:
##STR00010##
and a pharmaceutically acceptable excipient. In certain
embodiments, the compositions can further comprise an additional
chemotherapeutic agent.
[0016] Also provided are methods of treating cancer comprising
administration of a therapeutically effective amount of the
compound with the formula:
##STR00011##
to a subject in need thereof. In certain embodiments, the methods
can further comprise administration of an additional
chemotherapeutic agent.
[0017] Also provide herein are compounds having the formula:
##STR00012##
wherein A is C(O) or S(O).sub.2; wherein B is an alkyl, alkenyl, or
alkynyl; wherein n=0, 1, 2, or 3; wherein R.sub.1 is selected from
the group consisting of a halogen, hydrogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl, a
C.ident.C-aryl halide, and an aryl group; wherein R.sub.2 is
selected from the group consisting of hydrogen, alkyl, alkenyl, and
an aryl group; wherein R.sub.3 is selected from the group
consisting of hydrogen, alkyl, alkenyl, and an aryl group; and,
wherein R.sub.4 is selected from the group consisting of hydrogen,
alkyl, a compound of the formula:
##STR00013##
wherein B is alkenyl or alkynyl, R.sub.5 is selected from the group
consisting of hydrogen, halogen, and methoxy, and wherein each of
R.sub.6, R.sub.7, and R.sub.8 are independently selected from the
group consisting of hydrogen, bromine, chlorine, fluorine, keto,
hydroxyl, alkyl, alkenyl, alkoxy, an aminoalkenyl, and an
aminoalkoxy group with the proviso that at least one of R.sub.5,
R.sub.6, and R.sub.7 is not a methoxy group when A is C(O), n=0,
and R.sub.1, R.sub.2, and R.sub.3 are hydrogen, and with the
proviso that at least one of R.sub.6, R.sub.7, and R.sub.8 is not a
hydrogen, methoxy group, alkoxy, or aminoalkoxy group when R.sub.1,
R.sub.2, and R.sub.3 are hydrogen, and a compound of the
formula:
##STR00014##
wherein A is C(O) or S(O).sub.2, and is optionally joined to B by
X; wherein X, when present, is an alkyl, alkenyl, alkynyl, aryl, or
combination thereof; wherein n=0, 1, 2, or 3; wherein R.sub.9 is
selected from the group consisting of a halogen, hydrogen,
C.ident.C-alkyl, C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide,
C.ident.C-aryl, a C.ident.C-aryl halide, and an aryl group; wherein
R.sub.10 is selected from the group consisting of hydrogen, alkyl,
alkenyl, and an aryl group; and, wherein R.sub.11 is selected from
the group consisting of hydrogen, alkyl, alkenyl, and an aryl
group. In certain embodiments, compounds of the formula II wherein
R4 is independently selected from the group consisting of hydrogen,
alkyl, phenyl, and a compound of the formula:
##STR00015##
wherein A is C(O) or S(O).sub.2, and is optionally joined to B by
X, wherein X, when present, is an alkyl, alkenyl, alkynyl, aryl, or
combination thereof; wherein n=0, 1, 2, or 3; wherein R.sub.9 is
selected from the group consisting of a halogen, hydrogen,
C.ident.C-alkyl, C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide,
C.ident.C-aryl, a C.ident.C-aryl halide, and an aryl group; wherein
R.sub.10 is selected from the group consisting of hydrogen, alkyl,
alkenyl, and an aryl group; and, wherein R.sub.11 is selected from
the group consisting of hydrogen, alkyl, alkenyl, and an aryl group
are provided. In certain embodiments, R.sub.1 is a
C.ident.C-cycloalkyl group wherein said cycloalkyl is a C3 to C6
ring and/or wherein R.sub.3 is hydrogen or a thienyl group. In
certain embodiments, R.sub.1 is a C.ident.C-cycloalkyl, wherein
said cycloakyl is substituted at the ring carbon that is bound to
the alkynyl carbon with a hydroxyl group and/or wherein R.sub.3 is
hydrogen or a thienyl group. In certain embodiments, R.sub.1 is a
C.ident.C-phenyl or a C.ident.C-phenyl halide wherein the halide is
substituted ortho or para to the phenyl ring carbon that is bound
to the alkynyl carbon and/or wherein R.sub.3 is hydrogen or a
thienyl group. In certain embodiments where R.sub.4 is a compound
of formula IV, R.sub.1 and R.sub.9 are independently selected from
a group consisting of hydrogen, a halogen, and a
C.ident.C-cycloalkyl group wherein said cycloalkyl is a C3 to C6
ring and/or wherein R.sub.3 and R.sub.11 are independently selected
from a group consisting of hydrogen and a thienyl group or,
alternatively, R.sub.1 and R.sub.9 are independently selected from
the group consisting of hydrogen, a halogen, and a
C.ident.C-cycloalkyl group, wherein said cycloakyl is substituted
at the ring carbon that is bound to the alkynyl carbon with a
hydroxyl group and/or wherein R.sub.3 is hydrogen or a thienyl
group. In certain embodiments where R.sub.4 is a compound of
formula IV, R.sub.1 and R.sub.9 are independently selected from the
group consisting of hydrogen, a halogen, and is a C.ident.C-phenyl
or a C.ident.C-phenyl halide wherein the halide is substituted
ortho or para to the phenyl ring carbon that is bound to the
alkynyl carbon and/or wherein R.sub.3 is hydrogen or a thienyl
group. Also provided are compounds having a formula selected from
the group consisting of:
##STR00016## ##STR00017## ##STR00018##
Also provided are compounds of the formula:
##STR00019##
wherein R.sub.1 is either: (i) halogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl, a
C.ident.C-aryl halide, or an aryl group; or, (ii) an alkyl,
thioalkyl, aminoalkyl, or alkenyl group that is optionally fused to
R.sub.2 when R.sub.2 is alkyl and wherein the ring formed by the
fusion consists of 5, 6, 7, or 8 members; wherein R.sub.2 is alkyl;
wherein R.sub.3 is alkyl; wherein R.sub.4 is selected from the
group consisting of hydrogen, halogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl, a
C.ident.C-aryl halide, and an aryl group; and, wherein R.sub.5 is
selected from the group consisting of hydrogen, alkyl,
S(O).sub.2--R.sub.6, and C(O)--R.sub.6, wherein R.sub.6 is alkyl,
alkenyl, or alkynyl. In certain embodiments, the compound has the
structure:
##STR00020##
[0018] Also provided herewith are compositions comprising any of
the aforementioned compounds and a pharmaceutically acceptable
excipient. In certain embodiments, the composition can further
comprise an additional chemotherapeutic agent
[0019] Also provided herewith are methods of treating cancer that
comprise administration of a therapeutically effective amount of
any of the aforementioned compounds to a subject in need thereof.
In certain embodiments, the methods can further comprise
administration of an additional chemotherapeutic agent. Also
provided herein is the use of any of the aforementioned compounds
for treating cancer in a subject in need thereof. Also provided
herein is the use of any of the aforementioned compounds in the
manufacture of a medicament for treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The accompanying drawings, which are incorporated in and
form a part of the specification, illustrate certain embodiments of
the present invention. In the drawings:
[0021] FIG. 1A, B, and C illustrate A) Piperlongumine and its
cellular phenotypes (5); B) Convergent strategy for the synthesis
of PL analogs; and C) PL reacts with small-molecule thiols at C3
under neutral conditions, reaction time, 72 h.
[0022] FIG. 2 A, B, and C illustrate the contribution of PL's
electrophilic functionalities to cellular phenotypes. A)
Measurement of cellular ATP as a surrogate of viability
(CellTiter-Glo) and B) cellular reactive oxygen species levels
(CM-H2DCF-DA) in two cell lines. Data are expressed as mean.+-.SD
for four (CTG) or three (ROS) independent experiments. C)
Representative fluorescence microscopy images of HeLa cells treated
for 1 h with 20M PL, PL-H2, or PL-2,3H2.
[0023] FIG. 3 illustrates that oligomerization of PL leads to
greatly elevated potency for elevation of ROS and cell death in two
cell lines. A) aminoalkoxy C12 substituted monomer of PL (PL-MON),
dimer (PL-DI), and trimer (PLTRI). B) ATP levels and C) ROS levels
in HeLa and H1703 cells after 48 h (ATP) or 1.5 h (ROS) treatment
with the indicated concentrations of oligomer. Data are expressed
as mean.+-.SD for four (CTG) or three (ROS) independent
experiments.
[0024] FIG. 4 illustrates that certain piperlongumine analogs
decouple ROS elevation and cellular viability. A) ATP levels and B)
ROS levels in HeLa and H1703 cells after 48 h (ATP) or 1.5 h (ROS)
treatment with the indicated concentrations of analog. Data are
expressed as mean.+-.SD for four (CTG) or three (ROS) independent
experiments.
[0025] FIG. 5 illustrates that Piperlongumine's C7-C8 olefin is
unnecessary for depletion of cellular glutathione but essential for
protein glutathionylation. A) Levels of total cellular glutathione
were measured after 3-hour treatment of EJ cells with the indicated
concentrations of each analog. B) Quantification of protein
gluathionylation in HeLa cells after 6 h treatment, as detected by
immunofluorescence using a monoclonal antibody against glutathione.
Data are expressed as mean.+-.SD for three (A) or four (B)
independent experiments. C) Representative fluorescence microscopy
images showing protein glutathionylation following 6 h treatment
with either DMSO, PL, or PL-H2.
[0026] FIG. 6 illustrates that certain piperlongumine analogs show
selective toxicity toward transformed human fibroblasts (BJ-ELR).
Viability was measured by Crystal Violet staining after 48 h
treatment with A) PL-7, B) PL-SO2, C) PL-DIM, or D) PL-TRI. Data
are expressed as mean.+-.SD for three independent experiments.
[0027] FIG. 7 illustrates: A) Proposed non-limiting model for
PL-mediated protein glutathionylation of glutathione-binding
proteins (5). Note that the proposed protein glutathionylation does
not involve a direct reaction between the protein and glutathione.
Rather, the non-limiting model predicts that the glutathionylation
involves a linking PL molecule between the protein and glutathione.
B) Summary of the role of electrophilic functionalities of PL
analogs on cellular assay performance.
[0028] FIG. 8 A, B, C illustrates approaches to the synthesis of
diverse piperlongumine analogs. A) Synthesis of C4- and
C5-substituted analogs; B) Synthesis of C2-substituted analogs; C)
Synthesis of oligomeric piperlongumine analogs.
[0029] FIG. 9 illustrates reactivity of piperlongumine with methyl
thioglycolate, a thiol nucleophile.
[0030] FIG. 10 illustrates piperlongumine analogs elevate ROS
comparably with PL but show divergent outcomes in cell viability in
two additional cell lines (see also FIG. 4). Data are expressed as
mean.+-.SD for three independent experiments.
[0031] FIG. 11 illustrates comparable depletion of total cellular
glutathione by PL and two less toxic analogs in HeLa cells after 6
h compound treatment. Data are expressed as mean.+-.SD for three
independent experiments. Comparable data for the EJ cell line (FIG.
5) is shown for comparison.
[0032] FIG. 12 illustrates A) increased protein glutathionylation
is observed for both PL and glutathione disulfide (GSSG) by
immunofluorescence. Although treatment with the dithiol reducing
agent DTT is able to reverse the signal increase observed for GSSG,
no change in signal is observed for PL-treated wells following DTT
treatment. B) Quantitation of IF images. Data are expressed as
mean.+-.SD for three independent experiments. C) Protein
glutathionylation occurs within minutes of PL treatment. Data are
expressed as mean.+-.SD for three independent experiments.
[0033] FIG. 13 A, B illustrate results of an initial screen for
selective toxicity with PL and 8 analogs in the BJ/BJ-ELR model of
tumorigenesis. Viability was measured using crystal violet.
[0034] FIG. 14A, B, C illustrate the performance of PL and analogs
in the BJ/BJ-ELR model tumorigenesis. A) PL performance. B)
Bright-field images confirming selective loss of viability at
stated doses. C) Performance of analogs as shown in FIG. 6. Data
are expressed as mean.+-.SD for three independent experiments.
[0035] FIG. 15 illustrates the identification of probe compounds
that decouple ROS elevation and toxicity.
[0036] FIG. 16A, B, C, D illustrate compounds that relate the
observed toxicity to protein glutathionylation.
[0037] FIG. 17 illustrates that oligomers of piperlongumine
potently elevate ROS levels and decrease cell viability.
[0038] FIG. 18 illustrates selective cell death induced by improved
piperlongumine analogs in cancer cells. Normal cell lines: 184B5,
TIG3TD; Cancer cell lines: EJ, H1703, Hela; assays performed in a
12 well plate with crystal violet.
[0039] FIG. 19 illustrates selective cell death induced by improved
piperlongumine analogs in cancer cells. Piperlongumine analogs
selectively induced cell death in oncogenically transformed human
BJ skin fibroblasts (BJ vs BJELR) and human mammary epithelial
cells (HMEL vs HMELR). Isogenic non-transformed and transformed
cell line pairs: BJ vs BJELRas HMEL vs HMELRas. Assays performed in
a 12 well plate with crystal violet. "Dimer" and "Trimer" compounds
are as shown in FIG. 18.
DESCRIPTION
Definitions
[0040] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains. In the
case of inconsistencies between the present disclosure and the
issued patents, applications, and references that are cited herein,
the present disclosure will prevail. The nomenclature used to
describe organic radicals, e.g., hydrocarbons and substituted
hydrocarbons, generally follows standard nomenclature known in the
art, unless otherwise specifically defined.
[0041] As used herein, the term "salt(s)", refer to acidic salts
formed with inorganic and/or organic acids, as well as basic salts
formed with inorganic and/or organic bases. In addition, when a
compound contains both a basic moiety, such as, but not limited to,
a pyridine or imidazole, and an acidic moiety, such as, but not
limited to, a carboxylic acid, zwitterions ("inner salts") may be
formed and are included within the term "salt(s)" as used
herein.
[0042] As used herein the term "pharmaceutically acceptable salt",
is intended to include nontoxic, physiologically acceptable salts
synthesized from a compound which contains a basic or acidic
moiety.
[0043] As used herein the term "prodrug", refers to a compound that
is a drug precursor which, upon administration to a subject,
undergoes chemical conversion by metabolic and/or chemical
processes to yield an active compound or a salt and/or solvate
thereof. Prodrugs include acid derivatives well known to
practitioners of the art, such as, for example, esters prepared by
reaction of the parent acidic compound with a suitable alcohol, or
amides prepared by reaction of the parent acid compound with a
suitable amine.
[0044] As used herein, the term "solvate", means a physical
association of a compound with one or more solvent molecules,
whether organic or inorganic. This physical association involves
varying degrees of ionic and covalent bonding, including hydrogen
bonding. In certain instances the solvate will be capable of
isolation, for example when one or more solvent molecules are
incorporated in the crystal lattice of the crystalline solid.
"Solvate" encompasses both solution-phase and isolated solvates.
Non-limiting examples of suitable solvates include hydrates,
ethanolates, methanolates, isopropanolates and the like. "Hydrate"
is a solvate wherein the solvent molecule is water (H2O).
[0045] As used herein, the term "subject", refers to both human and
non-human mammals.
[0046] As used herein, the phrase "therapeutically effective
amount", refers to an amount of a compound which, when administered
to a subject in need thereof, is sufficient to cause any beneficial
change in any symptom or marker associated with cancer.
[0047] As used herein, the terms "alkyl", "alkenyl", "alkoxy",
"aminoalkyl", "aminoalkenyl", "aminoalkoxy", "cycloalkyl", "aryl",
and "phenyl" refer to both substituted and unsubstituted alkyl,
alkenyl, alkoxy, aminoalkyl, aminoalkenyl, aminoalkoxy, cycloalkyl,
aryl, and phenyl groups. The terms "alkyl", "alkenyl", "alkoxy",
"aminoalkyl", "aminoalkenyl", "aminoalkoxy", "cycloalkyl", "aryl",
and "phenyl" as used herein also refer to both branched and
unbranched alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl,
aminoalkoxy, cycloalkyl, aryl, and phenyl groups.
[0048] As used herein, the term "alkoxy" refers to C1-C6
alkoxys.
[0049] As used herein in the context of a chain, the term "alkoxy"
refers to both C1-C6 alkoxys and chains comprising repeating C1-C6
alkoxy subunits. In certain embodiments, an alkoxy chain can thus
be a polymethoxy, polyethoxy, polypropoxy, or polybutoxy chain.
[0050] As used herein, the term "aryl" refers to both homocyclic
and heterocyclic aryls.
[0051] As used herein, the term "halogen" refers to chlorine,
bromine, fluorine, or iodide.
[0052] As used herein, the term "substituted" refers to replacement
of one or more hydrogen atoms on a given group with one or more of
a cyano, hydroxyl, hydroxyalkyl, nitro, halogen, amino, carboxyl,
or --CO--NH2 group.
[0053] As used herein, the phrase "pharmaceutically acceptable"
refers to those compounds, materials, compositions, and/or dosage
forms which are suitable for use in contact with the tissues of
subjects without excessive toxicity, irritation, allergic response,
or other problem or complication commensurate with a reasonable
risk/benefit ratio.
iPLA Compounds
[0054] In certain embodiments, iPLA compounds provided herein that
comprise oligomers containing PL monomers, iPLA monomers, and/or
PLA monomers are referred to herein as "iPLA oligomers". Such iPLA
oligomers can exhibit marked increases in cancer cell toxicity
relative to their constituent monomers. Also provided herein are
iPLA compounds that comprise iPLA monomers of the instant
invention. In general, such iPLA oligomers can comprise any
combination of PL, PLA, and/or iPLA monomers. In certain
embodiments, iPLA oligomers can contain 2-3,2-4, 2-5, or 2-6 PL,
iPLA, and/or PLA monomers. However, preferred embodiments contain
2-3 PL and/or PLA monomers and more preferred embodiments contain 2
PL, iPLA, and/or PLA monomers. Also provided herein are
pharmaceutically acceptable salts, solvates, and prodrugs of iPLA
compounds and thus iPLA oligomers.
[0055] Multimerization of PL and/or PLA monomers can be
accomplished by a covalent linkage between any one of carbons 11,
12, or 13 (C11, C12, or C13) in the carbon chain of a PL and/or PLA
monomer. Numbering of the carbons of the PL and PLA carbon chain is
shown in FIG. 1A, where carbons 11, 12, and 13 correspond to
carbons of the phenyl ring of PL that are substituted with methoxyl
groups in PL. Without seeking to be limited by theory, it is
believed that any of C11, C12, or C13 carbons of the PL or PLA
monomer can serve as a linkage site as it is shown herein that the
methoxy groups that are linked to C11, C12, or C13 can be replaced
by other substituents without substantially altering biological
activity of the monomer (i.e. elevation of ROS or cellular
toxicity). In certain embodiments, monomers can be joined at either
the same carbon or at distinct carbons in the backbone of the PL or
PLA carbon chain. Thus, iPLA oligomers can, in certain embodiments,
comprise monomers that are both linked at C11, C12, or C13 (i.e. a
C11 to C11, a C12 to C12, or a C13 to C13 linkage). Oligomers where
the monomers are both linked at C12 of their respective carbon
chains are also provided. In other embodiments, PL and/or PLA
monomers are linked via distinct carbons in their backbones (i.e.
C11 to C12, C11 to C13, or C12 to C13) in an iPLA oligomer.
[0056] Covalent linkage of PL and/or PLA monomers in an iPLA
oligomer can be effected by a variety of molecules referred to
herein as "chains". Such chains can be either branched or
unbranched. Chain lengths include, but are not limited to chains of
any one of 2, 3, 4, 5, 6, 7, or 8 atoms to any one of 9, 10, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 atoms in
length. Such chains can also be substituted or unsubstituted. In
certain embodiments, the chain can comprise a branched or
unbranched alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl, and/or
an aminoalkoxy group that can be substituted or unsubstituted. In
certain embodiments, the chain can be joined to the C11, C12, or
C13 carbons of the PL or PLA monomer by groups comprising a part of
the chain that include, but are not limited to, an ether, ester, a
thioester, a thioether, an amide, or a keto group. In still other
embodiments, the chain can comprise a branched or unbranched alkyl,
alkenyl, alkoxy, aminoalkyl, aminoalkenyl, and/or aminoalkoxy group
that can be substituted or unsubstituted, where at least one of
said groups are joined in said chain via an ether, ester,
thioester, thioether, amide, --CH.sub.2--, or a keto group. Also
provided are embodiments where the chain comprises a branched or
unbranched alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl, or an
aminoalkoxy group that can be substituted or unsubstituted is
joined by an ether, ester, a thioester, a thioether, an amide,
--CH.sub.2--, or a keto group to one or more of a branched or
unbranched alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl, or an
aminoalkoxy group that can be substituted or unsubstituted. In
certain embodiments, chains that can be used to link PL or PLA
monomers include, but are not limited to, any of the following
compounds that can be branched and/or substituted, where X and Y
are independently selected from a group consisting of an ether,
ester, thioester, thioether, amide, --CH.sub.2--, and a keto group:
[0057] a)
--X--(CH.sub.2).sub.n1--N(R.sub.7)--(CH.sub.2).sub.n2--Y--, where
n.sub.1=1-8, n.sub.2=1-8, and R.sub.7 is selected from the group
consisting of hydrogen, alkyl, alkenyl, alkoxy, aminoalkyl,
aminoalkenyl, aminoalkoxy, and an aminoalkoxy group; [0058] b)
--X--(CH.sub.2).sub.n1--N(R.sub.7)--(CH.sub.2).sub.n2--Y--, where
n.sub.1=1-8, n.sub.2=1-8, and R.sub.7 is selected from the group
consisting of alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl,
aminoalkoxy, and the R.sub.7 group is linked to a third PL monomer
or to a third PLA monomer; [0059] c)
--X--(CH.sub.2).sub.n1--O--(CH.sub.2).sub.n2--Y--, wherein
n.sub.1=1-8, n.sub.2=1-8; [0060] d)
--X--(CH.sub.2).sub.n1--C(O)--O--(CH.sub.2).sub.n2--Y--, wherein
n.sub.1=1-8, n.sub.2=1-8; [0061] e)
--X--(CH.sub.2).sub.n1--S(O)--(CH.sub.2).sub.n2--Y--, wherein
n.sub.1=1-8, n.sub.2=1-8; [0062] f)
--X--(CH.sub.2).sub.n1--S--(CH.sub.2).sub.n2--Y--, wherein
n.sub.1=1-8, n.sub.2=1-8; [0063] g)
--X--(CH.sub.2).sub.n1--C(O)--N(R.sub.8)--(CH.sub.2).sub.n2--Y--,
wherein n.sub.1=1-8, n.sub.2=1-8, and R.sub.8 is selected from the
group consisting of hydrogen, alkyl, alkenyl, alkoxy, aminoalkyl,
aminoalkenyl, aminoalkoxy, and an aminoalkoxy group;
[0064] h)
X--(CH.sub.2).sub.n1--C(O)--N(R.sub.8)--(CH.sub.2).sub.n2--Y--,
wherein n.sub.1=1-8, n.sub.2=1-8, and R.sub.8 is selected from the
group consisting of alkyl, alkenyl, alkoxy, aminoalkyl,
aminoalkenyl, aminoalkoxy, and the aminoalkoxy group said R.sub.7
group is linked to a third PL monomer or to a third PLA monomer;
[0065] i) X--(CH.sub.2O).sub.n3--Y--, wherein n.sub.3=1-8, and,
[0066] j) --X--(CH.sub.2)n1-C(O)--(CH.sub.2)n2-Y--, wherein n1=1-8,
n2=1-8.
[0067] Oligomers comprising PLA monomers are also provided herein.
In the broadest sense, any PLA monomer disclosed herein may be
multimerized or combined with PL to yield an iPLA oligomer.
[0068] In certain embodiments, the PLA monomer will be of the
Formula I:
##STR00021##
[0069] where A is C(O) or S(O).sub.2; where n=0, 1, 2, or 3; where
the ortho-carbon of the phenyl ring is unsubstituted or substituted
with a halogen; where R.sub.1 is selected from the group consisting
of hydrogen, halogen, C.ident.C-alkyl, C.ident.C-cycloalkyl,
C.ident.C-cycloakyl halide, C.ident.C-aryl, C.ident.C-aryl halide,
and an aryl group; where R.sub.2 is selected from the group
consisting of hydrogen, alkyl, alkenyl, and an aryl group; where
R.sub.3 is selected from the group consisting of hydrogen, alkyl,
alkenyl, and an aryl group; and, where each of R.sub.4, R.sub.5,
and R.sub.6 is independently selected from the group consisting of
hydrogen, bromine, chlorine, fluorine, keto, hydroxyl, alkyl,
alkenyl, alkoxy, aminoalkyl, aminoalkenyl, and an aminoalkoxy
group, with the proviso that said compound is not a PL monomer.
Such PLA monomers can be linked to one another or to a PL monomer
via a covalent chain between any one of the C11, C12, or C13
groups. In certain embodiments, any one of the R.sub.4, R.sub.5,
and R.sub.6 groups of one PLA monomer can be linked to any one of
the R.sub.4, R.sub.5, and R.sub.6 groups of another PLA monomer,
where the PLA monomers are either identical or distinct. In certain
embodiments, any one of the R.sub.4, R.sub.5, and R.sub.6 groups of
one PLA monomer can be linked to any one of the C11, C12, or C13
groups of a PLA monomer. In certain embodiments, R.sub.1 is a
halogen in the PLA monomer. In certain embodiments, R.sub.1 is a
sulfur containing heteroaryl group in in the PLA monomer. In
certain embodiments, R.sub.1 is a thiophene group in the in the PLA
monomer.
[0070] In certain embodiments, the PLA monomer used in the iPLA
oligomer can comprise or consist of the compound of Formula I where
n=1, 2, 3. Such iPLA oligomers containing "ring expanded" PLA
monomers are anticipated to provide for improved selective toxicity
towards cancer cells relative to toxicity observed in non-cancer
cells. Ring expanded analogs comprising or consisting of the
compound of Formula I where n=1, 2, 3 and where any of the
corresponding R.sub.1-R.sub.6 positions shown in Formula I are
substituted with the corresponding Formula I R.sub.1-R.sub.6 groups
can be used as PLA monomers in iPLA oligomers. In certain
embodiments, such ring expanded analogs can consist of a
substituted derivative of the compound of Formula II, where the
ortho-carbon of the phenyl ring is substituted with a halogen. In
certain embodiments, such ring expanded analogs can consist of a
substituted derivative of the compound of Formula II, where the
ortho-carbon of the phenyl ring is substituted with a fluorine. In
certain embodiments, such ring expanded analogs can consist of a
substituted derivative of the compound of Formula II, where the
saturated carbon immediately adjacent to the nitrogen of the
cycloheptenimide ring (i.e the R.sub.1 group in the compound of
Formula I) is substituted with a heteroaryl group containing a
sulfur heteroatom. In certain embodiments, the saturated carbon
immediately adjacent to the nitrogen of the cycloheptenimide ring
(i.e the R.sub.1 group in the compound of Formula I) is substituted
with a 5 membered heteroaryl group having a sulfur heteroatom. In
certain embodiments, A is a S(O).sub.2 in the ring expanded analog.
In certain embodiments, R.sub.1 is a halogen in the ring expanded
analog. In certain embodiments, R.sub.1 is a sulfur containing
heteroaryl group in the ring expanded analog. In certain
embodiments, R.sub.1 is a sulfur containing pentacyclic heteroaryl
group in the ring expanded analog. In certain embodiments, n is 1
in the ring expanded analog.
[0071] In certain embodiments, the PLA monomer used in the iPLA
oligomer can comprise ring expanded cycloheptenimide analogs. An
exemplary ring expanded cycloheptenimide analog can comprise an
unsubstituted or substituted derivative of the compound with the
formula:
##STR00022##
[0072] Certain iPLA compounds provided herein comprise
unmultimerized PLA monomers. Such iPLA compounds are referred to
herein as "iPLA monomers" and can exhibit improved cancer cell
toxicity in their monomeric form relative to PL. The iPLA monomers
can thus be used in either their monomeric or multimeric forms in
methods and compositions provided herein. Also provided herein are
pharmaceutically acceptable salts, solvates, and prodrugs of iPLA
monomers.
[0073] In general, iPLA monomers can in certain embodiments be
compounds where an alkynyl, halogen, or aryl group is substituted
at the R.sub.1 position of compounds of Formula I. Such alkynl
groups at the R.sub.1 position can also be linked to a variety of
substituted or unsubstituted alkyl, cycloakyl, or aryl groups.
Suitable substitutions for such alkyl, cycloakyl, or aryl groups
include, but are not limited to, hydroxyls and halogens. In certain
embodiments, the iPLA monomers can thus comprise compounds of
formula I, where A is C(O) or S(O).sub.2, where n=0, 1, 2, or 3;
where the ortho-carbon of the phenyl ring is unsubstituted or
substituted with a halogen, where R.sub.1 is selected from the
group consisting of a halogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl,
C.ident.C-aryl halide, and an aryl group, where R.sub.2 is selected
from the group consisting of hydrogen, alkyl, alkenyl, and an aryl
group, where R.sub.3 is selected from the group consisting of
hydrogen, alkyl, alkenyl, and an aryl group, and where each of
R.sub.4, R.sub.5, and R.sub.6 is independently selected from the
group consisting of hydrogen, bromine, chlorine, fluorine, keto,
hydroxyl, alkyl, alkenyl, alkoxy, aminoalkyl, aminoalkenyl, and an
aminoalkoxy group. In certain embodiments, n is 1 in iPLA monomers
where an alkynyl group is substituted at the R.sub.1 position of
compounds of Formula I. In certain embodiments, A is S(O).sub.2 in
iPLA monomers where an alkynyl group is substituted at the R.sub.1
position of compounds of Formula I. In certain embodiments, n is 1
and A is S(O).sub.2 in iPLA monomers where an alkynyl group is
substituted at the R.sub.1 position of compounds of Formula I. In
certain embodiments, R.sub.1 is a halogen in the iPLA monomer. In
certain embodiments, R.sub.1 is a sulfur containing heteroaryl
group in in the iPLA monomer. In certain embodiments, R.sub.1 is a
thiophene group in the in the iPLA monomer.
[0074] In certain embodiments, iPLA monomers can comprise or
consist of the compound of Formula I where n=1, 2, 3. Such "ring
expanded" iPLA monomers can provide for improved selective toxicity
towards cancer cells relative to toxicity observed in non-cancer
cells. Improved selectivity (i.e. "therapeutic index") associated
with such ring-expanded iPLA monomers can provide for methods of
treatment where increased dosages of the iPLA monomer can be
provided while minimizing undesirable side effects.
[0075] Ring expanded analogs comprising or consisting of the
compound of Formula I where n=1, 2, 3 and where any of the
corresponding R.sub.1-R.sub.6 positions shown in Formula I are
substituted with the corresponding Formula I R.sub.1-R.sub.6 groups
provided herein can also be used as iPLA monomers. In certain
embodiments, such ring expanded analogs can consist of a
substituted derivative of the compound of Formula I, where the
ortho-carbon of the phenyl ring is substituted with a halogen. In
certain embodiments, such ring expanded analogs can consist of a
substituted derivative of the compound of Formula I, where the
ortho-carbon of the phenyl ring is substituted with a fluorine. In
certain embodiments, such ring expanded analogs can consist of a
substituted derivative of the compound of Formula I, where the
R.sub.1 group in the compound of Formula I is substituted with a
heteroaryl group containing a sulfur heteroatom. In certain
embodiments, the saturated carbon immediately adjacent to the
nitrogen of the cycloheptenimide ring (i.e the R.sub.1 group in the
compound of Formula I) is substituted with a 5 membered heteroaryl
group having a sulfur heteroatom. In certain embodiments, A is a
S(O).sub.2 in the ring expanded analog of Formula I. In certain
embodiments, n is 1 in the ring expanded analog of Formula I. In
certain embodiments, R.sub.1 is a halogen in the ring expanded
analog. In certain embodiments, R.sub.1 is a sulfur containing
heteroaryl group in the ring expanded analog. In certain
embodiments, R.sub.1 is a thiophene group in the ring expanded
analog.
[0076] In certain embodiments, iPLA monomers can comprise or
consist of the compound of Formula III where n=1, 2, 3.
##STR00023##
where A is C(O) or S(O).sub.2, R.sub.1 is selected from the group
consisting of hydrogen, alkyl, alkenyl, and an aryl group, and
R.sub.2 is selected from the group consisting of hydrogen, alkyl,
alkenyl, and an aryl group. In certain embodiments, such ring
expanded analog can consist of a substituted derivative of the
compound of Formula III, where the ortho-carbon of the phenyl ring
is substituted with a halogen. In certain embodiments, such ring
expanded analogs can consist of a substituted derivative of the
compound of Formula III, where the ortho-carbon of the phenyl ring
is substituted with a fluorine. In certain embodiments, such ring
expanded analogs can consist of a substituted derivative of the
compound of Formula III, where the saturated carbon immediately
adjacent to the nitrogen of the cycloheptenimide ring (i.e the
R.sub.1 group in the compound of Formula I) is substituted with a
heteroaryl group containing a sulfur heteroatom. In certain
embodiments, the saturated carbon immediately adjacent to the
nitrogen of the cycloheptenimide ring (i.e the R.sub.1 group in the
compound of Formula I) is substituted with a 5 membered heteroaryl
group having a sulfur heteroatom. In certain embodiments, A is a
S(O).sub.2 in the ring expanded analog of Formula III. In certain
embodiments, n is 1 in the ring expanded analog of Formula III. A
compound of Formula III where n=1, A is a S(O).sub.2, and R.sub.1
is selected from the group consisting of hydrogen, alkyl, alkenyl,
and an aryl group, and R.sub.2 is selected from the group
consisting of hydrogen, alkyl, alkenyl, and an aryl group.
[0077] In certain embodiments, the iPLA monomer can comprise ring
expanded cycloheptenimide analogs. An exemplary ring expanded
cycloheptenimide analog can comprise an unsubstituted or
substituted derivative of the compound with the formula:
##STR00024##
In certain embodiments, such ring expanded cycloheptenimide analogs
can consist of a substituted derivative of the compound of Formula
II, where the ortho-carbon of the phenyl ring is substituted with a
halogen. In certain embodiments, such cycloheptenimide analogs can
consist of a substituted derivative of the compound of Formula II,
where the ortho-carbon of the phenyl ring is substituted with a
fluorine. Methods for Making iPLA Compounds
[0078] Methods for synthesizing both iPLA oligomers and iPLA
monomers of the invention are also provided herein.
[0079] In certain embodiments, iPLA monomers can be obtained by a
convergent synthetic scheme as follows:
##STR00025##
where constituent rings of the iPLA monomer comprising desired R
groups or comprising desired R group precursors are conjoined. One
skilled in the art will also recognize that this scheme could also
be used for "C" compounds where n=3 to obtain compounds of Formula
(I) where n=3.
[0080] A two-step procedure for obtaining iPLA monomers where an
R.sub.1 of a compound of Formula I comprising an alkynyl group is
illustrated by the following exemplary reactions:
##STR00026##
[0081] Although the precursor compound of Formula I used in these
reactions contains an unsubstituted ortho carbon on the phenyl ring
and a methoxy group at R.sub.4, R.sub.5, and R.sub.6, other
precursor compounds containing any of an unsubstituted or
substituted ortho carbon on the phenyl ring and/or a group other
than hydrogen at any one of R.sub.2 or R.sub.3 and/or a group other
than methoxy group at any one or more of R.sub.4, R.sub.5, and/or
R.sub.6 can be used to obtain a desired iPLA monomer with an
alkynyl group at R.sub.1.
[0082] To synthesize iPLA oligomers, it is typically useful to
first synthesize a PLA or iPLA precursor monomer containing a
desired group at any one of R.sub.4, R.sub.5, and/or R.sub.6. A
procedure for obtaining a useful iPLA precursor is illustrated by
the following exemplary reaction:
##STR00027##
[0083] Although the precursor compound of Formula I used in this
exemplary reaction contains an unsubstituted ortho carbon on the
phenyl ring, hydrogens at R.sub.1, R.sub.2, and R.sub.3, and a
methoxy group at R.sub.4 and R.sub.6, other precursor compounds
containing any of an unsubstituted or substituted ortho carbon on
the phenyl ring and/or groups other than hydrogen at any one or
more of R.sub.1, R.sub.2, and R.sub.3, and/or a group other than
methoxy group at any one or more of R.sub.4 and/or R.sub.6, can be
used to obtain a desired iPLA monomer with a hydroxy group at
R.sub.5.
[0084] An iPLA monomer precursor where a hydroxyl precursor group
is of R.sub.4, R.sub.5, and/or R.sub.6 can also be obtained by the
convergent synthetic techniques described herein. Such iPLA monomer
precursors can be used to obtain iPLA oligomers where PL, PLA
and/or iPLA monomers are joined at any one of C11, C12, or C13.
[0085] Synthesis of useful iPLA oligomers with a PLA or iPLA
precursor is illustrated by the following exemplary reactions:
##STR00028## ##STR00029##
[0086] Again, such reactions can be performed with other precursor
compounds containing any of an unsubstituted or substituted ortho
carbon on the phenyl ring and/or groups other than hydrogen at any
one or more of R.sub.1, R.sub.2, and R.sub.3, and/or a group other
than methoxy group at any one or more of R.sub.4 and/or R.sub.6, to
obtain a desired iPLA oligomer.
[0087] The synthesis of additional PLA and iPLA monomer precursors
useful for synthesis of iPLA oligomers is also shown in the
following exemplary reactions
##STR00030##
[0088] It is anticipated that PLA and iPLA monomeric precursors
comprising ethoxy chains with terminal carboxyl groups can be
multimerized with other PLA and iPLA monomeric precursors
containing suitable reactive groups. Again, such reactions can be
performed with other precursor compounds containing any of an
unsubstituted or substituted ortho carbon on the phenyl ring and/or
groups other than hydrogen at any one or more of R.sub.1, R.sub.2,
and R.sub.3, and/or a group other than methoxy group at any one or
more of R.sub.4 and/or R.sub.6, to obtain a desired iPLA monomer
precursor for synthesis of a desired iPLA oligomer.
[0089] It is further contemplated that other synthetic methods for
obtaining certain iPLA oligomers and iPLA monomers described in the
Examples provided herein can be adapted by those skilled in the art
to provide for the synthesis of other iPLA oligomers and iPLA
monomers disclosed herein.
Other Compounds of the Invention and Methods of Making the Same
[0090] Also provided herein are various compounds of the
formula:
##STR00031##
[0091] wherein A is C(O) or S(O).sub.2;
[0092] wherein B is an alkenyl or alkynyl;
[0093] wherein Y is alkyl, alkenyl, alkynyl, aryl, or a combination
thereof,
[0094] wherein n=0, 1, 2, or 3;
[0095] wherein R.sub.1 is selected from the group consisting of a
halogen, hydrogen, C.ident.C-alkyl, C.ident.C-cycloalkyl,
C.ident.C-cycloakyl halide, C.ident.C-aryl, a C.ident.C-aryl
halide, and an aryl group;
[0096] wherein R.sub.2 is selected from the group consisting of
hydrogen, alkyl, alkenyl, and an aryl group;
[0097] wherein R.sub.3 is selected from the group consisting of
hydrogen, alkyl, alkenyl, and an aryl group;
[0098] wherein R.sub.9 is selected from the group consisting of a
halogen, hydrogen, C.ident.C-alkyl, C.ident.C-cycloalkyl,
C.ident.C-cycloakyl halide, C.ident.C-aryl, a C.ident.C-aryl
halide, and an aryl group;
[0099] wherein R.sub.10 is selected from the group consisting of
hydrogen, alkyl, alkenyl, and an aryl group; and,
[0100] wherein R.sub.11 is selected from the group consisting of
hydrogen, alkyl, alkenyl, and an aryl group.
[0101] An exemplary reaction that can provide for such compounds is
shown below:
##STR00032##
where A is C(O) and Y is alkyl, alkenyl, alkynyl, aryl, or a
combination thereof.
[0102] To accomplish such reactions, to the solution of the
heterocyclic reactants in THF at about -78.degree. C. is added
solution of n-BuLi in hexanes and stirred for about 15 minutes. To
this solution is added the compound
##STR00033##
and the reaction is stirred at about -78.degree. C. for about 3
hours. The reaction mixture is diluted with ethyl acetate, quenched
with aqueous ammonium chloride, extracted with EtOAc, washed with
brine, dried with anhydrous sodium sulfate and purified by column
chromatography using hexanes-ethyl acetate gradient (0 to 80%
EtOAc), to yield the product.
[0103] Also provided herein are compounds of the structure:
##STR00034##
wherein R.sub.1 is either: (i) halogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl, a
C.ident.C-aryl halide, or an aryl group; or, (ii) an alkyl,
thioalkyl, aminoalkyl, or alkenyl group that is optionally fused to
R.sub.2 when R.sub.2 is alkyl and wherein the ring formed by the
fusion consists of 5, 6, 7, or 8 members; wherein R.sub.2 is alkyl;
wherein R.sub.3 is alkyl; wherein R.sub.4 is selected from the
group consisting of hydrogen, halogen, C.ident.C-alkyl,
C.ident.C-cycloalkyl, C.ident.C-cycloakyl halide, C.ident.C-aryl, a
C.ident.C-aryl halide, and an aryl group; and, wherein R.sub.5 is
selected from the group consisting of hydrogen, alkyl,
S(O).sub.2--R.sub.6, and C(O)--R.sub.6, wherein R.sub.6 is alkyl,
alkenyl, or alkynyl.
[0104] Such compounds can be made by using suitable precursor
compounds in the manner disclosed herein for synthesis of
(E)-N-methacryloylbut-2-enamide:
##STR00035##
Pharmaceutical Compositions Comprising iPLA and Other Compounds of
the Invention
[0105] In practicing any of the methods of the present invention
involving administration of cancer inhibitory, preventative, or
mitigating agents to a subject, it is contemplated that a variety
of pharmaceutical compositions comprising active iPLA compounds or
other active compounds provided herein can be administered by a
variety of techniques. Such pharmaceutical compositions may be
formulated in various ways known in the art for administration
purposes. To prepare pharmaceutical compositions, a therapeutically
effective amount of an active iPLA compound, or a salt, solvate, or
prodrug thereof, is combined with one or more pharmaceutically
acceptable carriers and/or delivery vehicles. The active
ingredient, i.e., iPLA compound, in such compositions typically
comprises from about 0.1 percent by weight to about 99.9 percent by
weight of the composition, and often comprises from about 5 percent
by weight to about 95 percent by weight. Numerous pharmaceutically
acceptable carriers and delivery vehicles exist that are readily
accessible and well known in the art. Non-limiting illustrative
examples of pharmaceutically acceptable carriers and delivery
vehicles include aluminum stearate, lecithin, serum proteins such
as human serum albumin, buffer substances such as the various
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, and zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene,
polyoxypropylene-block polymers, polyethylene glycol and wool fat,
and the like.
[0106] The pharmaceutical compositions described herein may further
be prepared in a form suitable for administration orally,
percutaneously, by parenteral injection (including subcutaneous,
intramuscular, intravenous and intradermal), topically,
intranasally, by inhalation, or for application to a medical
device, such as an implant, catheter, or other device. Oral
administration or administration by injection are generally
preferred. In preparing compositions that permit oral delivery of
liquid preparations such as suspensions, syrups, elixirs and
solutions, any of the pharmaceutically acceptable carriers known in
the art may be used such as but not limited to, water, glycols,
oils, alcohols and the like. When solid pharmaceutically acceptable
carriers are desired, such as those that permit oral or rectal
administration; starches, sugars, kaolin, lubricants, binders,
cellulose and its derivatives, and disintegrating agents and any
other pharmaceutically acceptable carriers known in the art may be
used to prepare, for example, powders, pills, capsules and tablets.
For pharmaceutically acceptable compositions and preparations that
permit parenteral administration, the pharmaceutically acceptable
carriers often comprise sterile water, which may be supplemented
with various solutes to, for example, increase solubility.
Injectable solutions may be prepared in which the pharmaceutically
acceptable carrier comprises saline solution, glucose solution, or
a mixture thereof, which may include certain well-known
anti-oxidants, buffers, bacteriostats, and other solutes that
render the formulation isotonic with the blood of the intended
patient. The preparation of pharmaceutically acceptable
formulations is described in. e.g., Remington: The Science and
Practice of Pharmacy, 20.sup.th Ed., ed. A. Gennaro, Lippincott
Williams & Wilkins, 2000.
[0107] Active iPLA compounds provided herein in the compositions
may be used to treat cancer in combination with one another, or
with at least one additional biologically active agent.
Non-limiting illustrative examples of biologically active compounds
or agents that can be combined with iPLA compounds in compositions
provided herein include, but are not limited to, additional
chemotherapeutic agents. Such additional chemotherapeutic agents
include, but are not limited to, alkylating agents (including but
not limited to cyclophosphamide, mechlorethamine, chlorambucil,
melphalan), anthracyclines (including, but are not limited, to
daunorubicin, doxorubicin, epirubicin, idarubicin mitoxantrone,
valrubicin), cytoskeletal disruptors (including but are not limited
to taxanes such as paclitaxel and docetaxel), epothilones, histone
deacetylase inhibitors (including but not limited to vorinostat,
romidepsin), topoisomerase ii inhibitors (including but not limited
to etoposide, teniposide, tafluposide), kinase inhibitors
(including but are not limited to bortezomib, erlotinib, gefitinib,
imatinib, vismodegib), monoclonal antibodies (including but not
limited to bevacizumab, cetuximab, ipilimumab, ofatumumab,
ocrelizumab, panitumab, rituximab), nucleotide analogs and
precursor analogs (including but are not limited to azacytidine,
azathioprine, capecitabine, cytarabine, doxifluridine,
fluorouracil, gemcitabine, hydroxyurea, mercaptopurine,
methotrexate, and thioguanine), peptide antibiotics (including but
not limited to bleomycin, actinomycin), platinum-based agents
(including but not limited to carboplatin, cisplatin, oxaliplatin),
retinoids (tretinoin, alitretinoin, bexarotene) and vinca alkaloids
and derivatives (including but not limited to vinblastine,
vincristine, vindesine, and vinorelbine). In certain embodiments,
the additional chemotherapeutic agent can comprise another
compound, antibody, or protein that potentiates and/or relieves the
side effects of anti-cancer drugs. In certain embodiments, such
additional chemotherapeutic agent thus include, but are not limited
to, anti-angiogenesis, anti-nausea agents, and the like. In certain
embodiments, such additional chemotherapeutic agent thus include,
but are not limited to agents such as erythropoietin and the
like.
[0108] It is further contemplated that the pharmaceutical
compositions provided herein can include agents that promote uptake
of iPLA compounds by target cells or regions of interest in a
subject or a patient. Such target cells and regions of interest
include, but are not limited to, cancer cells, tumors, and their
metastases. Agents that promote uptake of iPLA compounds, include
but are not limited to, a variety of organic and/or amphiphilic
compounds. In certain embodiments, the agent can comprise DMSO,
PEG, phospholipids, fatty acids, and combinations thereof. In
certain embodiments, the composition can comprise liposomes or
micelles that contain the iPLA compound and facilitate uptake by
target cells or regions of interest in a subject or a patient. In
certain embodiments, the composition can comprise nanoparticles
that facilitate uptake of the iPLA compound by target cells or
regions of interest in a subject or a patient. Conjugates of iPLA
compounds to any of the aforementioned agents are also provided
herein.
Methods of Treating Cancer
[0109] Certain aspects of the current disclosure provide methods
for treating cancer. As used herein, treatment of cancer is
understood to embrace methods whereby establishment, progression,
recurrence, or spread of at least one of a malignant growth, tumor,
solid tumor, or its metastases are inhibited, delayed, arrested, or
otherwise controlled in a subject. Such subjects can be mammals
susceptible to cancer that include, but are not limited to, humans,
companion animals (dogs, cats, and the like), and livestock
(horses, cows, sheep, pigs, and the like).
[0110] Cancers that can be treated by the iPLA compounds,
compositions, and methods provided herein include, but are not
limited to, cancers of major organ systems and their metastases.
Treatment of cancers including, but not limited to, cancers of the
brain, breast, thyroid, blood, skin, lung, liver, pancreas, colon,
prostate, endometrium, cervix, ovaries, larynx, oropharynx,
esophagus, bladder, and their metastases are thus provided
herein.
[0111] Administration of a combination of one or more of the iPLA
compounds provided herein and one or more additional
chemotherapeutic agents is also contemplated. Administration of a
combination can be sequential, wherein treatment with one agent is
done before treatment with a second agent. Alternatively,
administration can be concurrent where treatment with two or more
agents occurs at the same time. Sequential administration can be
done within a reasonable time after the completion of a first
therapy before beginning a second therapy. Administration of
multiple agents concurrently can be in the same daily dose or in
separate doses.
[0112] In certain embodiments, the additional chemotherapeutic
agent can comprise another compound, antibody, or protein that is
an anti-cancer agent. Such anti-cancer agents include, but are not
limited to, alkylating agents (including, but not limited to,
cyclophosphamide, mechlorethamine, chlorambucil, melphalan),
anthracyclines (including, but are not limited to, daunorubicin,
doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin),
cytoskeletal disruptors (including, but are not limited to, taxanes
such as paclitaxel and docetaxel), epothilones, histone deacetylase
inhibitors (including, but not limited to, vorinostat, romidepsin),
topoisomerase II inhibitors (including, but not limited to,
etoposide, teniposide, tafluposide), kinase Inhibitors (including,
but are not limited to, bortezomib, erlotinib, gefitinib, imatinib,
vismodegib), monoclonal antibodies (including but not limited to
bevacizumab, cetuximab, ipilimumab, ofatumumab, ocrelizumab,
panitumab, rituximab), nucleotide analogs and precursor analogs
(including, but are not limited to, azacytidine, azathioprine,
capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine,
hydroxyurea, mercaptopurine, methotrexate, and thioguanine),
peptide antibiotics (including, but not limited to, bleomycin,
actinomycin), platinum-based agents (including, but not limited to,
carboplatin, cisplatin, oxaliplatin), retinoids (tretinoin,
alitretinoin, bexarotene) and vinca alkaloids and derivatives
(including, but not limited to, vinblastine, vincristine,
vindesine, and vinorelbine).
[0113] In certain embodiments, the additional chemotherapeutic
agent can comprise another compound, antibody, or protein that
potentiates and/or relieves the side effects of anti-cancer drugs.
In certain embodiments, such additional chemotherapeutic agent thus
includes, but are not limited to, anti-angiogenesis, anti-nausea
agents, and the like. In certain embodiments, such additional
chemotherapeutic agents include, but are not limited to, agents
such as erythropoietin and the like.
[0114] The pharmaceutical compositions of the present invention may
be formulated into a variety of dosage forms depending upon the
particular composition contemplated. Likewise, a variety of modes
of administration are possible depending upon the particular
composition and dosage form. In certain embodiments, administration
is by intravenous injection. In certain embodiments, administration
is by delivery to a location of interest. Locations of interest
include, but are not limited to, tumors and their metastases or
locations of one or more cancer cells. Delivery to a location of
interest can be effected by any method that provides for directed
introduction of the iPLA compound or a composition comprising the
same to a site in a subject or patient and include, but are not
limited to, direct injection, delivery by a cathether, delivery by
a stereotactically guided instrument, implantation of a drug
delivery device, implantation of a device or substance that
provides for release of the iPLA compound, and the like.
Impregnation of devices and or substances with iPLA compounds or
compositions to provide for release at a location of interest is
thus provided herein. In certain embodiments, a location of
interest can be a site from which a tumor, cancer cells, or other
cancerous growth have been resected or otherwise ablated or
removed. In such cases, any of the aforementioned delivery methods,
devices, or substances can be used to provide the iPLA compound or
composition at the location of interest to prevent or delay
recurrence. Exemplary substances that are useful for localized
delivery of anti-cancer agents include, but are not limited to,
various poly(ester-carbonate)-collagen and/or poly(carbonate
ester)s comprising 6-hydroxyhexanoic acid monomers that are
disclosed by Wolinsky et al. J Control Release. 2010 Jun. 15;
144(3):280-7; Liu et al. Ann Surg Oncol. 2010 April; 17(4):1203-13;
and Wolinsky et al., Macromolecules, 2007, 40, 7065-7068.
Therapeutically effective amounts of an active compound, its salts,
prodrugs, solvates, a pharmaceutical composition thereof, or a
combination therapy will depend on absorption, distribution,
metabolism, and excretion rates of the components of the therapy.
Dosage values will also vary with the severity of the condition to
be alleviated. It is further understood that for any particular
subject, specific dosage regimens and schedules may be adjusted
over time according to the individual's need and the professional
judgment of the person administering or supervising the
administration. The therapeutically effective amount of the
inhibitory compound administered will be determined empirically,
and will also be based on considerations such as the particular
inhibitor or combination used, the age, sex, diet, body weight, and
general health status of the individual, the treatment effect
desired, administration route, the severity and course of the
disease, and the like. It is expected that in certain embodiments,
the typical therapeutically effective dose range will be from about
0.1 mg/kg to about 50 mg/kg per dose, which can be given one to
several times per day, or alternatively as a continuous infusion.
Such administration can be used as a chronic or acute therapy. In
still other embodiments, a therapeutically effective dose can range
from about 0.1, 0.2, 0.3, 0.4, or 0.5 mg/kg to any one of about
1.0, 2.0, 2.3, 2, 4, 2.5, 3.0, 5.0, 10, 15, 20, 25, 30, 40, 50 or
100 mg/kg. In still other embodiments, a therapeutically effective
dose can range from about 0.1, 0.2, 0.3, 0.4, 0.5, 2.0, 2.3, 2, 4,
or 2.5 mg/kg to any one of about 3.0, 5.0, 10, 15, 20, 25, 30, 40,
50 or 100 mg/kg. In still other embodiments, a therapeutically
effective dose can range from about 0.2 mg/kg to about 2.4 mg/kg or
about 0.2 mg/kg to about 5 mg/kg. In still other embodiments, a
therapeutically effective dose can range from about 2.5 mg/kg to
about 50 mg/kg or about 100 mg/kg. When the compositions comprise a
combination of an iPLA compound and one or more additional
biologically active agent(s), both the compound and the additional
agent(s) are usually present at dosage levels of between about 10
to 100%, and more preferably between about 10 and 80% of the dosage
normally administered in a monotherapy regimen.
[0115] In certain embodiments, therapeutically effective amounts of
compounds and/or compositions provided herein can be determined
and/or adjusted by any of a variety of biological markers of
cancer. In certain embodiments, the therapeutically effective
amounts can be determined and/or adjusted by monitoring the levels
of certain metabolites, proteins and/or nucleic acids in a subject.
In certain embodiments, the DNA methylation status of the subject
or patient can serve as a useful cancer biomarker to determine
and/or adjust therapeutically effective amounts of compounds and/or
compositions provided herein.
Kit for Treating Cancer
[0116] In certain embodiments contemplated herein, kits comprising
at least one pharmaceutical composition of an iPLA compound or
combination of iPLA compounds and one or more pharmaceutically
acceptable carriers, as well as one or more containers are
provided.
[0117] The composition(s) of the kit that comprise an iPLA compound
may be provided in any form. Composition forms provided in the kit
can include, but are not limited to, tablets, capsules, pills,
liquid solutions or dried powders. In certain embodiments where the
composition(s) are provided in a liquid solution, such liquid
solution can be for example an aqueous solution. When the
composition(s) provided are a dry powder, the powder can be
reconstituted by the addition of a suitable solvent, which can also
be provided.
[0118] The container will generally include a vial into which the
pharmaceutical composition may be placed, and preferably suitably
aliquotted. The kits of the present invention will also typically
include a means for containing the composition(s) in a container in
close confinement for commercial sale. Such containers may include
injection or blow-molded plastic containers into which the desired
vials are retained.
[0119] The kit can also comprise a device or a component of a
device for performing the methods provided herein. Devices, or
components of devices, include, but are not limited to, syringes
and other implements useful for delivery of the composition to the
blood stream or a specific organ, e.g. the liver. In certain
embodiments, compositions comprising an iPLA compound can be
provided in unit dose form. In addition or in the alternative, the
kits can provide an instructional material which describes
performance of one or more methods for treatment of cancer that are
provided herein, or a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration. Instructions can also be provided as a fixed, fully
detachable, or partially detachable label that is associated with
one or more containers in the kit. The instructions associated with
the kit can provide directions for preparing the pharmaceutical
composition for administration and/or instructions for
administration of the iPLA compound containing pharmaceutical
composition to a subject in need thereof.
EXAMPLES
[0120] The following disclosed embodiments are merely
representative of the invention, which may be embodied in various
forms. It is anticipated that methods disclosed herein for the
synthesis of certain compounds can be adapted to provide for the
synthesis of other compounds disclosed or claimed herein. Thus,
specific structural and functional details disclosed herein are not
to be interpreted as limiting.
Example 1
Synthesis of Exemplary iPLA Compounds
[0121] Synthesis of piperlongumine analogs. Piperlongumine analogs
were in general synthesized by a convergent strategy that entailed
coupling commercially available or synthetically accessible lactams
and carboxylic acid chlorides (FIG. 1B). Additional analogs bearing
substituents at C2 were generated by selective iodination of PL at
C2 and palladium catalyzed cross-coupling (for complete synthesis
details, see FIG. S1).
General Methods:
[0122] Dry solvents (anhydrous THF, CH.sub.2Cl.sub.2 and toluene
etc.) were purchased from Sigma-Aldrich. Unless otherwise stated,
all reagents were obtained from commercial sources and used without
further purification. Infrared spectra were recorded on a Nicolet
Avatar 370 DTGS FTIR. 1H NMR spectra were recorded on Varian
Unity/Inova 500 (500 MHz), or Bruker Ultrashield 300 (300 MHz)
spectrometers. .sup.1H NMR data are reported as follows: chemical
shift in parts per million relative to CHCl.sub.3 (7.26 ppm),
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broadened), coupling constant (Hz), and integration.
13C NMR spectra were recorded on Varian Unity/Inova 500 (125 MHz)
or Bruker Ultrashield 300 (75 MHz) spectrometers. .sup.13C NMR
chemical shifts are reported in parts per million relative to
solvent. All .sup.13C NMR spectra were determined with broadband
decoupling. High-resolution mass spectra (HRMS) were obtained
through the Broad Institute Chemical Biology Analytical Chemistry
facility. All reactions were magnetically stirred and monitored by
thin-layer chromatography (TLC) using E. Merck silica gel 60 F254
precoated plates (0.25 mm) Flash chromatography was performed
either on EM Science silica gel 60 (230-400 mesh) or using a
CombiFlash companion system (Teledyne ISCO, Inc.) with pre-packed
FLASH silica gel columns (Biotage, Inc.).
Experimental Procedures and Spectra Data
##STR00036##
[0124] Synthesis of non-commercially available acid chloride: To a
solution of acids (A, 0.1-0.2 M) in dry CH.sub.2Cl.sub.2 was added
oxalyl chloride (5.0 equiv., 2.0 M in CH.sub.2Cl.sub.2) and
catalytic amount of DMF (0.01 equiv.). The reaction mixture was
stirred at room temperature for 2-5 hours before the solvent was
removed. The residue was dried under high vacuum, then used to the
next step without any further purification.
Amide Formation:
[0125] Method a: To a solution of compound C (0.1 M, 1.2 equiv.) in
dry THF in a flame-dried Schlenk flask was added 1.6 M solution of
n-BuLi (1.3 equiv.) dropwise at -78.degree. C. under nitrogen
atmosphere. After 15 min, a solution of the corresponding acid
chloride B (1.0 equiv.) in dry THF was added dropwise. After 10
min, the reaction was gradually warmed up to room temperature and
the solvent was evaporated under vacuum. The residue was purified
by flash chromatography to provide the desired amide D. Method b:
To a solution of acid chloride B (1.0 equiv., 0.1 M) in
CH.sub.2Cl.sub.2 was added triethylamine (TEA, 3.0 equiv.) and
compound C (1.2 equiv.). The reaction mixture was stirred at room
temperature for overnight before it was quenched with saturated
aqueous NH4Cl, and extracted with CH.sub.2Cl.sub.2 (3 times). The
combined organic phases were washed with brine and dried over
MgSO.sub.4. After filtration and concentration, the residue was
purified by flash chromatography provide the desired amide D.
##STR00037##
1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]piperidin-2-one
[0126] Method a; Yield: 95%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.64 (d, J=15.6 Hz, 1H), 7.36 (d, J=15.5 Hz, 1H), 6.78 (s,
2H), 3.88 (s, 6H), 3.87 (s, 3H), 3.80 (m, 2H), 2.60 (m, 2H), 1.88
(m, 4H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 173.9, 169.5,
153.4, 143.5, 140.2, 130.7, 121.3, 105.5, 60.9, 56.2, 44.6, 34.9,
22.6, 20.6; IR (thin film, cm.sup.-1) 2941, 2839, 1671, 1614, 1579,
1503, 1454, 1416, 1348, 1386, 1315, 1267, 1242, 1196, 1152, 1121,
1174, 1002, 969, 915, 888, 825, 730, 775, 700, 675, 595, 527; m/z
found: 320.56 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.17H.sub.21NO.sub.5: 319.1420; found 319.1419.
##STR00038##
1-(3-(3,4,5-trimethoxyphenyl)propanoyl)piperidin-2-one
[0127] Method a; Yield: 64%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 6.44 (s, 2H), 3.83 (s, 6H), 3.80 (s, 3H), 3.70 (m, 2H),
3.21 (t, J=6.0 Hz, 2H), 2.89 (t, J=6.0 Hz, 2H), 2.51 (m, 2H), 1.80
(m, 4H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 176.4, 173.7,
153.3, 137.3, 136.4, 105.6, 61.1, 56.3, 44.3, 41.6, 35.2, 31.8,
22.7, 20.5; IR (thin film, cm.sup.-1) 2939, 1688, 1588, 1508, 1457,
1421, 1365, 1343, 1291, 1238, 1194, 1153, 1125, 1005, 827, 581; m/z
found: 323.31 [M+H.sup.+]; HRMS (FAB) calcd For
C.sub.17H.sub.23NO.sub.5:321.1576; found 321.1568.
##STR00039##
4-methyl-1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-5,6-dihydropyrid-
in-2(1H)-one
[0128] Method a; Yield: 52%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.66 (d, J=15.6 Hz, 1H), 7.44 (d, J=15.6 Hz, 1H), 6.80 (s,
2H), 5.84 (s, 1H), 4.02 (t, J=6.3 Hz, 2H), 3.89 (s, 6H), 3.87 (s,
3H), 2.40 (t, J=6.3 Hz, 2H), 2.03 (s, 3H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.7, 166.0, 157.7, 153.3, 143.4, 139.9,
130.7, 121.3, 121.2, 105.5, 60.9, 56.1, 41.5, 29.9, 22.9; IR (thin
film, cm.sup.-1) 2939, 1679, 1644, 1580, 1613, 1504, 1464, 1386,
1417, 1352, 1277, 1241, 1318, 1214, 1123, 1182, 1153, 1068, 1105,
1002, 1049, 1019, 971, 913, 862, 778, 824, 791, 643; m/z found:
332.07 [M+H.sup.+]; HRMS (FAB) calcd for C.sub.18H.sub.21NO.sub.5:
331.1420; found 331.1420.
##STR00040##
1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-3,6-dihydropyridin-2
(1H)-one
[0129] Method b; Yield: 42%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.60 (d, J=15.6 Hz, 1H), 7.42 (d, J=15.3 Hz, 1H), 6.79 (s,
2H), 5.88 (m, 1H), 5.83 (m, 1H), 4.36 (m, 2H), 3.89 (s, 6H), 3.87
(s, 3H), 3.19 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
171.0, 169.1, 153.4, 144.0, 136.5, 130.6, 122.1, 121.4, 121.0,
105.6, 61.0, 56.2, 45.8, 35.1; IR (thin film, cm.sup.-1) 2939,
2839, 1687, 1580, 1613, 1504, 1418, 1383, 1462, 1397, 1349, 1316,
1272, 1244, 1154, 1183, 1124, 1003, 944, 825, 676, 589; m/z found:
317.64 [M+H.sup.+]; HRMS (FAB) calcd for C.sub.17H.sub.19NO.sub.5:
317.1263; found 317.1261.
##STR00041##
3,4-dimethyl-1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-5,6-dihydrop-
yridin-2(1H)-one
[0130] Method a; Yield: 46%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.65 (d, J=15.6 Hz, 1H), 7.42 (d, J=15.6 Hz, 1H), 6.80 (s,
2H), 3.95 (t, J=6.3 Hz, 2H), 3.89 (s, 6H), 3.87 (s, 3H), 2.41 (t,
J=6.3 Hz, 2H), 1.99 (s, 3H), 1.91 (s, 3H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 169.1, 167.0, 153.3, 150.0, 143.0, 139.9,
130.8, 125.8, 121.4, 105.5, 60.8, 56.1, 41.0, 31.1, 20.6, 12.2; IR
(thin film, cm.sup.-1) 2936, 2838, 1666, 1615, 1579, 1503, 1453,
1417, 1390, 1353, 1316, 1279, 1208, 1256, 1175, 1153, 1067, 1122,
1096, 1002, 981, 895, 824, 730, 782, 758, 701, 671, 619, 593; m/z
found: 345.92 [M.sup.+]; HRMS (FAB) calcd for
C.sub.19H.sub.23NO.sub.5: 345.1576; found 345.1574.
##STR00042##
4-methoxy-1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-1,5-dihydro-2H--
pyrrol-2-one
[0131] Method a; Yield: 32%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.94 (d, J=15.9 Hz, 1H), 7.77 (d, J=15.9 Hz, 1H), 6.85 (s,
2H), 5.15 (s, 1H), 4.38 (s, 2H), 3.90 (s, 9H), 3.88 (s, 3H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 176.2, 170.2, 164.8,
153.4, 145.2, 140.3, 130.5, 118.7, 105.7, 94.8, 60.9, 58.7, 56.2,
48.7; IR (thin film, cm.sup.-1) 3099, 2947, 1720, 1665, 1622, 1581,
1506, 1470, 1449, 1420, 1435, 1327, 1368, 1311, 1279, 1181, 1252,
1192, 1125, 1155, 1046, 971, 992, 913, 842, 819, 666; m/z found:
334.37 [M+H.sup.+]; HRMS (FAB) calcd for C.sub.17H.sub.19NO.sub.6:
333.1212; found 333.1211.
##STR00043##
3-ethyl-4-methyl-1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-1,5-dihy-
dro-2H-pyrrol-2-one
[0132] Method a; Yield: 30%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 8.01 (d, J=15.9 Hz, 1H), 7.81 (d, =15.9 Hz, 1H), 6.86 (s,
2H), 4.29 (s, 2H), 3.91 (s, 6H), 3.88 (s, 3H), 2.32 (q, J=7.5 Hz,
2H), 2.08 (s, 3H), 1.10 (t, J=7.5 Hz, 3H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 171.0, 165.0, 153.4, 150.9, 145.5, 140.1,
134.4, 130.5, 118.3, 105.7, 60.9, 56.2, 52.2, 16.8, 13.4, 12.8; IR
(thin film, cm.sup.-1) 2966, 2936, 1710, 1665, 1580, 1616, 1504,
1453, 1418, 1365, 1391, 1320, 1281, 1226, 1245, 1155, 1124, 1174,
1081, 1013, 934, 826, 801, 777, 729, 604; m/z found: 346.51
[M+H.sup.+]; FIRMS (FAB) calcd for C.sub.19H.sub.23NO.sub.5:
345.1576; found 345.1577.
##STR00044##
1-(3-(3,4,5-trimethoxyphenyl)propanoyl)-5,6-dihydropyridin-2(1H)-one
[0133] Method a; 86%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
6.89 (m, 1H), 6.47 (s, 2H), 6.00 (m, 1H), 3.97 (t, J=6.3 Hz, 2H),
3.84 (s, 6H), 3.81 (s, 3H), 3.25 (t, J=7.5 Hz, 2H), 2.93 (t, J=7.5
Hz, 2H), 2.40 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
175.4, 165.3, 153.0, 145.2, 136.8, 136.2, 125.8, 105.5, 60.7, 56.0,
40.9, 40.8, 31.5, 24.5; IR (cm.sup.-1) 2930, 1686, 1627, 1588,
1508, 1457, 1421, 1385, 1303, 1237, 1216, 1179, 1124, 1027, 1007,
818, 731, 587; m/z found: 320.26 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.17H.sub.21NO.sub.5: 319.1420; found 319.1421.
##STR00045##
1-[(2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-1,5,6,7-tetrahydro-2H-aze-
pin-2-one
[0134] Method a; Yield: 75%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.67 (d, J=15.6 Hz, 1H), 7.35 (d, J=15.6 Hz, 1H), 6.76 (s,
2H), 6.51 (m, 1H), 6.08 (d, J=11.7 Hz, 1H), 3.97 (t, J=6.0 Hz, 2H),
3.86 (s, 6H), 3.85 (s, 3H), 2.38 (q, J=6.3 Hz, 2H), 1.97 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 171.3, 167.7, 153.4,
144.2, 142.0, 140.1, 130.5, 128.0, 120.2, 105.6, 60.9, 56.2, 41.2,
26.1, 25.5; IR (thin film, cm.sup.-1) 2937, 1669, 1615, 1580, 1504,
1453, 1417, 1390, 1318, 1358, 1274, 1200, 1152, 1077, 1124, 1003,
926, 812, 730, 588; m/z found: 332.00 [M+H.sup.+]; HRMS (FAB) calcd
for C.sub.18H.sub.21NO.sub.5: 331.1420; found 331.1423.
##STR00046##
1-(1-benzothien-2-ylcarbonyl)-5,6-dihydropyridin-2(1H)-one
[0135] Method a; Yield: 60%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.82 (m, 3H), 7.39 (m, 2H), 6.96 (m, 1H), 6.04 (d, J=9.6
Hz, 1H), 3.96 (t, J=6.6 Hz, 2H), 2.58 (m, 2H); .sup.13C NMR (125
MHz, CDCl.sub.3) .delta. 167.8, 165.5, 145.6, 141.7, 138.4, 138.0,
129.7, 126.7, 125.6, 125.0, 124.7, 122.5, 44.1, 24.8; IR (thin
film, cm.sup.-1) 3057, 2889, 1659, 1593, 1559, 1516, 1423, 1458,
1469, 1383, 1334, 1227, 1184, 1159, 1128, 1086, 1011, 972, 904,
866, 819, 845, 801, 722, 636, 678; m/z found: 258.32 [M+H.sup.+];
HRMS (FAB) calcd for C.sub.14H.sub.11NO.sub.2S: 257.0510; found
257.0511.
##STR00047##
1-(1-benzofuran-2-ylcarbonyl)-5,6-dihydropyridin-2(1H)-one
[0136] Method a; Yield, 80%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.68 (d, J=7.8 Hz, 1H), 7.52 (d, J=8.4 Hz, 1H), 7.42 (m,
2H), 7.28 (m, 1H), 7.00 (m, 1H), 6.07 (d, J=9.6 Hz, 1H), 4.00 (t,
J=6.6 Hz, 2H), 2.61 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 164.9, 163.8, 155.0, 149.3, 145.6, 127.3, 127.1, 125.0,
123.5, 122.7, 113.0, 112.1, 43.4, 24.8; IR (cm.sup.-1) 3059, 2889,
2359, 1666, 1590, 1561, 1471, 1382, 1447, 1422, 1350, 1291, 1245,
1220, 1178, 1128, 1111, 1048, 1019, 968, 939, 908, 885, 846, 814,
747, 702, 612; m/z found: 241.97 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.14H.sub.11NO.sub.3: 241.0739; found 241.0744.
##STR00048##
1-(3-methyl-1H-indene-2-carbonyl)-5,6-dihydropyridin-2(1H)-one
[0137] Method a; Yield, 13%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.43 (m, 2H), 7.33 (m, 21-1), 6.95 (dt, J=9.9, 3.9 Hz, 1H),
6.00 (d, J=9.9 Hz, 1H), 3.96 (t, J=6.6 Hz, 2H), 3.67 (q, J=2.1 Hz,
2H), 2.55 (m, 2H), 2.32 (q, J=2.1 Hz, 3H); HRMS (FAB) calcd for
C.sub.16H.sub.15NO.sub.2: 253.1103; found 253.1106.
##STR00049##
1-[(2Z)-2-fluoro-3-phenylprop-2-enoyl]-5,6-dihydropyridin-2(1H)-one
[0138] Method a; 38%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.63 (dd, J=8.1, 1.2 Hz, 2H), 7.36 (m, 3H), 6.94 (dt, J=9.9, 4.2
Hz, 1H), 6.69 (d, J=35.7 Hz, 1H), 6.02 (dt, J=9.9, 1.8 Hz, 1H),
3.88 (t, J=6.6 Hz, 2H), 2.55 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 166.1 (d, J=33.5 Hz, 1C), 164.5, 151.2 (d,
J=273.4 Hz, 1C), 145.9, 131.7 (d, J=4.0 Hz, 1C), 130.3 (d, J=7.8
Hz, 1C), 129.3 (d, J=2.6 Hz, 1C), 128.6, 124.6, 116.1 (d, J=5.3 Hz,
1C), 43.2, 24.6; IR (thin film, cm.sup.-1) 3057, 2892, 1698, 1678,
1650, 1493, 1470, 1449, 1422, 1383, 1344, 1306, 1231, 1212, 1197,
1108, 1049, 1019, 1001, 973, 954, 923, 910, 873, 835, 817, 773,
760, 692, 645; m/z found: 246.25 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.14H.sub.12FNO.sub.2: 245.0852; found 245.0853.
##STR00050##
1-(5,6,7-trimethoxy-3,4-dihydronaphthalene-2-carbonyl)-5,6-dihydropyridin-
-2(1H)-one
[0139] Method b; Yield, 66%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 6.91-6.95 (m, 2H), 6.55 (s, 1H), 5.99 (dt, J=9.6, 1.8 Hz,
1H), 3.89 (t, J=6.6 Hz, 2H), 3.89 (s, 3H), 3.84 (s, 3H), 3.82 (s,
3H), 2.85 (t, J=8.1 Hz, 2H), 2.50 (m, 4H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 174.0, 165.2, 151.5, 150.4, 145.2, 143.0,
134.8, 132.9, 128.4, 125.0, 122.6, 107.9, 60.8, 60.6, 55.9, 43.0,
24.6, 23.5, 20.5; HRMS (FAB) calcd for C.sub.19H.sub.21NO.sub.5:
343.1420; found 343.1415.
##STR00051##
1-(2-fluoro-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropyridin-2(-
1H)-one
[0140] Method a; Yield, 43%; Characterized as a 2/1 mixture with
trans as major; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.93-6.99
(m, 1H, major), 6.87 (s, 2H, major), 6.84-6.88 (m, 0.5H, minor),
6.64 (d, J=35.4 Hz, 1H, major), 6.57 (s, 1H, minor), 6.51 (d,
J=20.4 Hz, 0.5H, minor), 6.04 (dt, J=9.9, 1.8 Hz, 1H, major), 5.93
(dt, J=9.9, 1.8 Hz, 0.5H, minor), 3.80-3.90 (m, 2H (major), 1H
(minor)), 3.86 (s, 3H, major), 3.85 (s, 6H, major), 3.81 (s, 1.5H,
minor), 3.80 (s, 3H, minor), 2.50-2.68 (m, 2H, major), 2.33-2.40
(m, 1H, minor); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 166.1 (d,
J=33.3 Hz, 1C, major), 164.5 (major), 163.9 (d, J=36.2 Hz, 1C,
minor), 163.8 (minor), 153.2 (??), 152.7 (??), 152.5 (??), 149.6
(d, J=512.8 Hz, 1C, minor), 149.5 (d, J=536.3 Hz, 1C, major), 149.2
(minor), 148.9, (major), 139.3 (d, J=3.3 Hz, 1C), 138.2 (??), 127.0
(d, J=4.0 Hz, 1C), 126.8 (d, J=10.4 Hz, 1C), 124.6 (major), 124.4
(minor), 118.9 (d, J=3.9 Hz, 1C), 116.4 (d, J=5.0 Hz, 1C), 114.0
(d, J=23.55 Hz, 1C), 107.8, 107.7, 106.2 (d, J=2.7 Hz, 1C), 60.9,
56.1, 43.3, 41.8, 24.6, 24.2; HRMS (FAB) calcd for
C.sub.17H.sub.18FNO.sub.5: 335.1169; found 335.1171.
##STR00052##
(E)-1-(3-(3-bromophenyl)prop-2-enoyl)-5,6-dihydropyridin-2(1H)-one
[0141] Method a; Yield, 71%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.54 (s, 1H), 7.46 (d, J=15.6 Hz, 1H), 7.26-7.31 (m, 3H),
7.04-7.09 (m, 1H), 6.75-6.81 (m, 1H), 5.87 (dt, J=9.6, 1.8 Hz, 1H),
3.86 (t, J=6.6 Hz, 2H), 2.28-2.33 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.5, 165.8, 145.6, 141.6, 137.2, 132.8,
130.8, 130.2, 127.0, 125.7, 123.3, 122.9, 41.6, 24.8; HRMS (FAB)
calcd for C.sub.14H.sub.12BrNO.sub.2: 305.0051; found 305.0050.
##STR00053##
(E)-1-(3-(2-methoxyphenyl)prop-2-enoyl)-5,6-dihydropyridin-2(1H)-one
[0142] Method a; Yield, 35%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 8.09 (d, J=15.9 Hz, 1H), 7.58 (m, 2H), 7.32 (td, J=8.1, 1.5
Hz, 1H), 6.91 (m, 3H), 6.03 (dt, J=9.6, 1.8 Hz, 1H), 4.03 (t, J=6.3
Hz, 2H), 3.88 (s, 3H), 2.46 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 169.3, 165.7, 158.4, 145.2, 138.9, 131.2,
128.8, 125.9, 124.2, 122.1, 120.6, 111.1, 55.5, 41.6, 24.8; HRMS
(FAB) calcd for C.sub.15H.sub.15NO.sub.3: 257.1052; found
257.1053.
##STR00054##
(E)-1-(3-(4-methoxyphenyl)prop-2-enoyl)-5,6-dihydropyridin-2(1H)-one
[0143] Method a; Yield, 15%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.73 (d, J=15.6 Hz, 1H), 7.54 (d, J=8.7 Hz, 2H), 7.41 (d,
J=15.3 Hz, 1H), 6.94 (m, 1H), 6.89 (d, J=8.7 Hz, 2H), 6.04 (dt,
J=9.9, 1.8 Hz 1H), 4.03 (t, J=6.6 Hz, 2H), 3.83 (s, 3H), 2.46 (m,
2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.1, 165.8, 161.3,
145.2, 143.6, 130.0, 127.9, 126.0, 119.5, 114.5, 55.4, 41.6, 24.8;
IR (thin film, cm.sup.-1) 2932, 1683, 1600, 1573, 1512, 1466, 1387,
1422, 1333, 1304, 1289, 1255, 1216, 1173, 1136, 1114, 1051, 1032,
980, 913, 822, 610. m/z found: 258.11 [M+H.sup.+]; HRMS (FAB) calcd
for C.sub.15H.sub.15NO.sub.3: 257.1052; found 257.1053.
##STR00055##
(E)-4-(3-oxo-3-(2-oxo-5,6-dihydropyridin-1(2H)-yl)prop-1-enyl)benzaldehyd-
e
[0144] Method a; Yield, 15%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 10.02 (s, 1H), 7.88 (d, J=8.7 Hz, 2H), 7.70 (m, 3H), 7.58
(d, J=15.6 Hz, 1H), 6.97 (m, 1H), 6.05 (dt, J=9.6 Hz, 1.8 Hz, 1H),
4.05 (t, J=6.6 Hz, 2H), 2.50 (m, 2H); IR (thin film, cm.sup.-1)
2940, 2839, 1698, 1681, 1579, 1505, 1453, 1418, 1385, 1343, 1313,
1278, 1225, 1158, 1121, 1049, 1020, 1000, 938, 910, 870, 818, 778,
762, 731, 703, 644, 615, 560; m/z found: 256.27 [M+H.sup.+]; HRMS
(FAB) calcd for C.sub.15H.sub.13NO.sub.3: 255.0895; found
255.0901.
##STR00056##
1-[(2E)-3-(2-chlorophenyl)prop-2-enoyl]-5,6-dihydropyridin-2(1H)-one
[0145] Method a; Yield: 53%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 8.15 (d, J=15.6 Hz, 1H), 7.73 (m, 1H), 7.50 (d, J=15.6 Hz,
1H), 7.42 (m, 1H), 7.29 (m, 3H), 6.98 (m, 1H), 6.07 (dt, J=9.6, 1.8
Hz, 1H), 4.08 (t, J=6.6 Hz, 2H), 2.51 (m, 2H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 168.5, 165.8, 145.6, 139.1, 135.0, 133.4,
130.7, 130.0, 128.0, 127.0, 125.8, 124.4, 41.6, 24.8; IR (thin
film, cm.sup.-1) 2934, 1677, 1616, 1470, 1385, 1330, 1298, 1216,
1185, 1135, 1054, 1038, 972, 818, 758; m/z found: 262.09
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.14H.sub.12C.sub.1NO.sub.2:
261.0557; found 261.0560.
##STR00057##
1-[(2E)-3-phenylprop-2-enoyl]-5,6-dihydropyridin-2(1H)-one
[0146] Method b; Yield 40%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.75 (d, J=15.6 Hz, 1H), 7.56-7.58 (m, 2H), 7.50 (d, J=15.6
Hz, 1H), 7.34-7.37 (m, 3H), 6.90-6.96 (m, 1H), 6.03 (dt, J=9.9, 1.5
Hz, 1H), 4.03 (t, J=6.6 Hz, 2H), 2.43-2.49 (m, 2H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 168.9, 165.7, 145.4, 143.5, 135.1,
130.0, 128.7, 128.3, 125.8, 121.9, 41.4, 24.7; HRMS (FAB) calcd for
C.sub.14H.sub.13NO.sub.2: 227.0946; found 227.0946.
##STR00058##
(E)-1-(3-(4-fluorophenyl)prop-2-enoyl)-5,6-dihydropyridin-2(1H)-one
[0147] Method a; Yield 74%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.71 (d, J=15.6 Hz, 1H), 7.56 (m, 2H), 7.44 (d, J=15.6 Hz,
1H), 7.06 (m, 2H), 6.94 (m, 1H), 6.05 (dt, J=9.9, 1.8 Hz, 1H), 4.04
(t, J=6.6 Hz, 2H), 2.48 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 168.8, 165.8, 160.4 (d, J=259.0 Hz, 1C), 145.5, 142.3,
131.4 (d, J=3.5 Hz, 1C), 130.2 (d, J=8.2 Hz, 1C), 125.8, 121.7 (d,
J=2.3 Hz, 1C), 115.9 (d, J=21.5 Hz, 1C), 41.6, 24.8; IR (thin film,
cm.sup.-1) 2932, 1674, 1620, 1598, 1507, 1470, 1414, 1386, 1330,
1298, 1215, 1159, 1136, 1183, 1096, 1119, 1051, 977, 864, 820, 738,
648, 610, 570; m/z found: 246.24 [M+H+]; HRMS (FAB) calcd for
C14H12FNO2: 245.0852; found 254.0858.
##STR00059##
1-{(2E)-3-[3-(trifluoromethyl)phenyl]prop-2-enoyl}-5,6-dihydro
pyridin-2(1H)-one
[0148] Method a; Yield: 88%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.69-7.78 (m, 3H), 7.46-7.61 (m, 3H), 6.93-6:99 (m, 1H),
6.05 (dt, J=9.6, 1.8 Hz, 1H), 4.04 (t, J=6.6 Hz, 2H), 2.50 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.5, 165.8, 145.7,
141.3, 135.9, 131.3 (q, 2J=32.3 Hz, 1C), 131.2, 129.3, 126.3 (q,
3J=3.7 Hz, 1C), 125.6, 124.8 (q, 3'J=3.75 Hz, 1C), 123.8 (q,
1J=269.0 Hz, 1C), 123.7, 41.6, 24.7; IR (thin film, cm.sup.-1)
2917, 1683, 1623, 1471, 1438, 1387, 1335, 1299, 1270, 1249, 1217,
1185, 1165, 1121, 1096, 1076, 1051, 974, 911, 857, 822, 802, 693,
660, 578, 560; m/z found: 296.27 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.15H.sub.12F.sub.3NO.sub.2: 295.0820; found 295.0823.
##STR00060##
1-(2-naphthoyl)-5,6-dihydropyridin-2(1H)-one
[0149] Method a; Yield 87%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 8.16 (s, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.83 (m, 2H), 7.53
(m, 3H), 6.98 (m, 1H), 5.99 (dt, J=9.6, 1.8 Hz, 1H), 4.04 (t, J=6.3
Hz, 2H), 2.61 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
173.9, 165.4, 145.5, 134.8, 133.4, 132.5, 129.1, 127.7, 127.6,
127.5, 126.4, 125.3, 124.8, 43.4, 24.9; IR (thin film, cm.sup.-1)
3055, 2889, 1626, 1670, 1599, 1506, 1467, 1422, 1381, 1354, 1289,
1244, 1222, 1192, 1151, 1102, 1048, 1019, 968, 929, 911, 864, 815,
801, 775, 759, 731, 701, 665, 646, 621; m/z found: 252.20
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.16H.sub.13NO.sub.2:
251.0946; found 251.0954.
##STR00061##
(E)-1-(2-methyl-3-phenylprop-2-enoyl)-5,6-dihydropyridin-2(1H)-one
[0150] Method b; Yield 57%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.26-7.42 (m, 5H), 6.90-6.96 (m, 2H), 5.99 (dt, J=9.9, 1.8
Hz, 1H), 3.92 (t, J=6.3 Hz, 2H), 2.49-2.55 (m, 2H), 2.14 (d, J=1.5
Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 176.1, 165.0,
145.3, 136.0, 134.9, 132.8, 129.3, 128.1, 127.6, 125.1, 42.7, 24.6,
15.5; HRMS (FAB) calcd for C.sub.15H.sub.15NO.sub.2: 241.1103;
found 241.1107.
##STR00062##
(E)-1-(3-phenylbut-2-enoyl)-5,6-dihydropyridin-2(1H)-one
[0151] Method a; 10%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.56 (m, 2H), 7.37 (m, 3H), 6.92 (m, 2H), 6.02 (dt, J=9.9, 1.8 Hz,
1H), 4.00 (t, J=6.6 Hz, 2H), 2.51 (d, J=1.2 Hz, 3H), 2.46 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.0, 165.6, 151.6,
145.0, 142.7, 128.7, 128.4, 126.6, 125.9, 121.8, 41.1, 24.8, 18.6;
HRMS (FAB) calcd for C.sub.15H.sub.15NO.sub.2: 241.1103; found
241.1110.
##STR00063##
1-[(2-oxo-2H-chromen-3-yl)carbonyl]-5,6-dihydropyridin-2(1H)-one
[0152] Method a; Yield 86%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.91 (s, 1H), 7.55 (m, 2H), 7.26 (m, 2H), 6.98 (m, 1H),
5.96 (dt, J=9.6, 1.8 Hz, 1H), 4.06 (t, J=6.3 Hz, 2H), 2.57 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.3, 165.7, 158.4,
145.2, 138.9, 131.2, 128.9, 125.9, 124.2, 122.2, 120.6, 111.1,
55.5, 41.6, 24.8; IR (thin film, cm.sup.-1) 1726, 1691, 1671, 1609,
1570, 1457, 1386, 1318, 1265, 1229, 1130, 1011, 820, 730. m/z
found: 270.25 [M+H.sup.+]; HRMS (FAB) calcd For
C.sub.15H.sub.11N.sub.4: 269.0688; found 269.0687.
##STR00064##
1-{[(E)-2-phenylvinyl]sulfonyl}-5,6-dihydropyridin-2(1H)-one
[0153] Method a; Yield 57%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.59 (d, J=15.6 Hz, 1H), 7.43 (m, 2H), 7.33-7.35 (m, 3H),
7.17 (d, J=15.3 Hz, 1H), 6.75-6.82 (m, 1H), 5.90 (dt, J=9.6, 1.8
Hz, 1H), 3.90 (t, J=6.6 Hz, 2H), 2.44-2.50 (m, 2H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 163.8, 144.9, 144.1, 132.1, 131.3,
129.0, 128.6, 124.8, 124.3, 43.3, 25.1; IR (thin film, cm.sup.-1)
3061, 2923, 1682, 1612, 1576, 1470, 1449, 1381, 1350, 1287, 1239,
1155, 1132, 996, 863, 848, 817, 746, 672; HRMS (FAB) calcd for
C.sub.13H.sub.13NO.sub.3S: 263.0616; found 263.0623.
##STR00065##
General Procedure for the Synthesis of 5-substituted
dihydropyridone
[0154] An oven-dried, two-necked, round-bottomed flask is charged
with dodecylbenzenesulfonic acid (216 mg, 0.66 mmol) flushed with
nitrogen, and equipped with a rubber septum, and an argon inlet.
The flask is charged with 28% aqueous ammonia (13.2 mL) by syringe.
After the gas evolution ceases, the mixture is stirred at room
temperature to give a clear solution, then cooled to 0.degree. C.
in an ice-water bath.
2-Allyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.33 g, 7.93 mmol)
is added dropwise over 5 min via a syringe and the mixture is
stirred at room temperature for 30 min. One of the aldehydes (6.61
mmol) is added drop-wise via a syringe over 5 min at room
temperature. The slurry mixture is vigorously stirred at room
temperature for 6 h and transferred to a separatory funnel with
water (15 mL) and saturated aqueous NaCl solution (30 mL). The
aqueous layer is extracted three times with diethyl ether (30 mL),
and the combined organic layers were washed with saturated aqueous
NaCl solution (15 mL), dried over anhydrous MgSO.sub.4, filtered,
and concentrated under reduced pressure to afford crude amine,
which was purified by flash chromatography (isopropylamine 4.8% in
hexane/ethyl acetate from 100:0 to 90:10) to afford pure amine.
[0155] One of the amine products (11.3 mmol) was dissolved in dry
THF (55 mL); triethylamine (NEt.sub.3, 1.50 g, 16.93 mmol) and
N,N-dimethylpyridin-4-amine (DMAP, 140 mg, 1.13 mmol) were added.
The mixture was then cooled at 0.degree. C. and acryloyl chloride
(1.50 g, 16.9 mmol) dissolved in dry THF (55 mL) was added
drop-wise; a yellow/white precipitate formed immediately. The
reaction was stirred at 0.degree. C. for 30 min and then warmed up
to room temperature and stirred for another 2 hours. At the end,
the solvent was removed under reduced pressure; crude product was
subject to flash chromatography (hexane/ethyl acetate 100:0 to
70:30):
[0156] One of the intermediates (500 mg, 2.16 mmol) was dissolved
in dry CH.sub.2Cl.sub.2 (22 mL); Grubbs catalyst 2.sup.nd
generation (184 mg, 0.22 mmol) was added and the mixture was heated
at reflux. For the case where R=3-pyridyl, TFA (0.41 mL, 5.40 mmol)
was added. After 1 h the mixture was cooled to room temperature and
concentrate to dryness to give a brown oil. Purification by flash
chromatography (hexane/ethyl acetate=30/70 to 0/100 except for
R=3-pyridyl: only ethyl acetate) provides the desired lactam, which
was used in the amide formation step under either condition a or
b.
##STR00066##
(E)-6-phenyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropyridi-
n-2(1H)-one
[0157] Method a; Yield 88%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.61 (d, J=15.6 Hz, 1H), 7.44 (d, J=15.6 Hz, 1H), 7.13-7.26
(m, 5H), 6.80 (s, 2H), 6.63 (m, 1H), 6.01 (dd, J=9.9, 2.7 Hz, 1H),
5.94 (d, J=6.6 Hz, 1H), 3.82 (s, 6H), 3.81 (s, 3H), 2.97-3.06 (m,
1H), 2.76 (dd, J=18.6, 5.7 Hz, 1H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.5, 165.8, 153.2, 144.0, 142.4, 140.2,
140.0, 130.5, 128.4, 127.2, 125.9, 125.8, 120.8, 105.4, 60.8, 56.0,
54.2, 31.3; IR (thin film, cm.sup.-1) 1683, 1580, 1504, 1315, 1275,
1242, 1185, 1124, 908; 818, 749, 729, 697; m/z found: 394.45
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.23H.sub.23NO.sub.5:
393.1576; found 393.1583.
##STR00067##
(E)-6-(pyridin-3-yl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihyd-
ropyridin-2(1H)-one
[0158] Method a; Yield 54%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 8.58 (d, J=1.5 Hz, 1H), 8.52 (d, J=4.8 Hz, 1H), 7.69 (d,
J=15.6 Hz, 1H), 7.55 (d, J=8.1 Hz, 1H), 7.49 (d, J=15.5 Hz, 1H),
7.23-7.28 (m, 1H), 6.81 (s, 2H), 6.75 (d, J=7.8 Hz, 1H), 6.13 (dd,
J=9.6, 2.7 Hz, 1H), 6.05 (d, J=6.6 Hz, 1H), 3.90 (s, 6H), 3.89 (s,
3H), 3.12-3.18 (m, 1H), 2.82 (dd, J=18.6, 6.3 Hz, 1H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 168.5, 165.2, 153.4, 148.7, 147.7,
144.7, 142.1, 140.2, 135.9, 133.8, 130.4, 126.3, 123.3, 120.5,
105.6, 60.9, 56.2, 52.6, 30.9; IR (thin film, cm.sup.-1) 2938,
2839, 1683, 1614, 1579, 1503, 1454, 1418, 1388, 1349, 1314, 1275,
1243, 1188, 1154, 1123, 1039, 1004, 820, 733, 712; m/z found:
395.53 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.22H.sub.22N.sub.2O.sub.5: 394.1529; Found 394.1533.
##STR00068##
(E)-6-(4-methoxyphenyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-di-
hydropyridin-2(1H)-one
[0159] Method a; Yield 35%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.67 (d, J=15.6 Hz, 1H), 7.48 (d, J=15.5 Hz, 1H), 7.14 (d,
J=8.7 Hz, 2H), 6.83 (d, J=8.7 Hz, 2H), 6.80 (s, 2H), 6.70-6.75 (m,
1H), 6.08 (dd, J=9.6, 3.0 Hz, 1H), 5.97 (d, J=6.6 Hz, 1H), 3.89 (s,
6H), 3.88 (s, 3H), 3.76 (s, 3H), 3.01-3.08 (m, 1H), 2.79 (dd,
J=18.6, 6.6 Hz, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
168.5, 165.8, 158.8, 153.3, 144.0, 142.6, 140.0, 132.3, 130.6,
127.1, 126.0, 121.0, 113.8, 105.5, 60.8, 56.1, 55.2, 53.8, 31.4; IR
(thin film, cm.sup.-1) 2936, 1683, 1613, 1581, 1505, 1455, 1418,
1389, 1350, 1277, 1248, 1221, 1154, 1125, 1034, 824, 731; m/z
found: 424.36 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.24H.sub.25NO.sub.6: 423.1682; found 423.1689.
##STR00069##
(E)-6-cyclohexyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropy-
ridin-2(1H)-one
[0160] Method a; Yield 78%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.66 (d, J=15.6 Hz, 1H), 7.39 (d, J=15.3 Hz, 1H), 6.78 (s,
2H), 6.01-6.04 (m, 1H), 4.73 (m, 1H), 3.88 (s, 6H), 3.87 (s, 3H),
2.58 (m, 2H), 1.62-1.71 (m, 6H), 1.01-1.10 (m, 5H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 168.2, 166.0, 153.2, 143.7, 143.5,
139.8, 130.7, 125.6, 120.7, 105.4, 60.8, 56.0, 55.9, 40.0, 30.8,
29.4, 26.5, 26.00, 25.98, 25.95; IR (thin film, cm.sup.-1) 2927,
2850, 1681, 1614, 1580, 1504, 1417, 1449, 1389, 1347, 1316, 1241,
1274, 1183, 1124, 1153, 1033, 1004, 976, 912, 818, 778, 728, 647;
m/z found: 400.39 [M+H.sup.+]; FIRMS (FAB) calcd for
C.sub.23H.sub.29NO.sub.5: 399.2046; found 399.2054.
##STR00070##
(E)-6-cyclopentyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydrop-
yridin-2(1H)-one
[0161] Method a; Yield 72%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.66 (d, J=15.6 Hz, 1H), 7.39 (d, J=15.5 Hz, 1H), 6.81 (m,
1H), 6.78 (s, 2H), 6.05 (dd, J=9.9, 3.0 Hz, 1H), 4.85 (dd, J=10.2,
6.0 Hz, 1H), 3.88 (s, 6H), 3.86 (s, 3H), 2.63-2.73 (m, 1H), 2.45
(dd, J=18.6, 6.3 Hz, 1H), 2.18-2.30 (m, 1H), 1.15-1.70 (m, 8H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.1, 165.8, 153.3,
143.7, 143.4, 139.9, 130.7, 125.7, 120.7, 105.4, 60.8, 56.1, 55.3,
42.7, 30.7, 29.7, 28.5, 25.0, 24.9; IR (thin film, cm.sup.-1) 2944,
2870, 1684, 1615, 1581, 1505, 1454, 1418, 1389, 1366, 1351, 1318,
1276, 1244, 1195, 1154, 1127, 1006, 823; m/z found: 386.60
[M+H.sub.+]; HRMS (FAB) calcd for C.sub.22H.sub.27NO.sub.5:
385.1899; found 385.1894.
##STR00071##
(E)-6-(thiophen-2-yl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihy-
dropyridin-2(1H)-one
[0162] Method a; Yield 19%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.71 (d, J=15.6 Hz, 1H), 7.43 (d, J=15.6 Hz, 1H), 7.13 (d,
J=5.1 Hz, 1H), 6.84-6.95 (m, 3H), 6.79 (s, 2H), 6.26 (d, J=6.0 Hz,
1H), 6.08 (dd, J=9.9, 3.0 Hz, 1H), 3.87 (s, 6H), 3.86 (s, 3H),
2.98-3.07 (m, 1H), 2.86 (dd, J=18.6, 6.0 Hz, 1H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 168.2, 165.0, 153.4, 144.5, 143.4, 142.7,
140.2, 130.6, 126.5, 126.2, 125.8, 124.5, 120.7, 105.6, 60.9, 56.2,
50.9, 31.5; IR (cm.sup.-1) 2938, 2838, 1683, 1612, 1579, 1503,
1453, 1417, 1389, 1350, 1315, 1274, 1242, 1184, 1154, 1123, 1040,
1003, 913, 818, 778, 732, 703, 591; m/z found: 400.06 [M+H.sup.+];
HRMS (FAB) calcd for C.sub.21H.sub.21NO.sub.5S: 399.1140; found
399.1140.
##STR00072##
(E)-6-(thiophen-3-yl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihy-
dropyridin-2(1H)-one
[0163] Method a; Yield 53%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.63 (d, J=15.6 Hz, 1H), 7.38 (d, J=15.6 Hz, 1H), 7.17-7.19
(m, 1H), 6.98 (s, 1H), 6.92 (d, J=4.8 Hz, 1H), 6.73 (s, 2H),
6.68-6.73 (m, 1H), 5.96-6.03 (m, 2H), 3.81 (s, 6H), 3.80 (s, 3H),
2.85-2.98 (m, 1H), 2.75 (dd, J=18.6, 6.0 Hz, 1H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 168.3, 165.5, 153.3, 144.2, 142.8, 141.3,
140.1, 130.5, 126.3, 126.1, 126.0, 121.7, 120.8, 105.5, 60.9, 56.1,
51.2, 30.7; IR (thin film, cm.sup.-1) 3102, 2937, 2838, 1732, 1682,
1613, 1579, 1503, 1453, 1417, 1384, 1354, 1314, 1274, 1242, 1183,
1152, 1122, 1042, 1002, 974, 923, 864, 816, 772, 732, 684, 638,
593; m/z found: 400.46 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.21H.sub.21NO.sub.5S: 399.1140; found 399.1152.
##STR00073##
(E)-5-(thiophen-2-yl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihy-
dropyridin-2(1H)-one
[0164] Method a; Yield 64%; .sup.1H NMR (300 MHz, CDCl.sub.3)
.delta. 7.67 (d, J=15.6 Hz, 1H), 7.43 (d, J=15.6 Hz, 1H), 7.23-7.27
(m, 1H), 6.93-7.03 (m, 3H), 6.81 (s, 2H), 6.15 (dd, J=9.6, 1.5 Hz,
1H), 4.34 (dd, J=18.6, 7.8 Hz, 1H), 4.11-4.18 (m, 2H), 3.90 (s,
6H), 3.88 (s, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.6,
165.2, 153.3, 147.1, 144.3, 140.8, 140.2, 130.5, 127.1, 125.5,
125.4, 125.0, 120.6, 105.6, 60.9, 56.2, 48.4, 36.4; m/z found:
400.03 [M+H.sup.+]; HRMS (FAB) calcd For C.sub.21H.sub.21NO.sub.5S:
399.1140; found 399.1144.
##STR00074##
[0165] Experiment Procedure:
[0166] Iodine (2.00 g, 7.89 mmol) was added to a solution of
piperlongumine (1.00 g, 3.15 mmol) in a 1:1 mixture of CCl.sub.4
and pyridine (32 mL) at room temperature. The mixture was stirred
overnight, wrapped, in an aluminium foil before saturated aqueous
NH.sub.4Cl (160 mL) was added. The mixture was extracted with ethyl
acetate (3.times.65 mL). The organic extract was dried over
MgSO.sub.4, filtered, and concentrated under vacuum. The crude
product was purified by flash chromatography (hexane/ethyl
acetate=80/20 to 50/50) to give the desired iodide in 98% yield.
.sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.69 (d, J=15.0 Hz, 1H),
7.67 (m, 1H), 7.39 (d, J=15.6 Hz, 1H), 6.80 (s, 2H), 4.09 (t, J=6.6
Hz, 2H), 3.90 (s, 6H), 3.88 (s, 3H), 2.50 (m, 2H); .sup.13C NMR
(125 MHz, CDCl.sub.3) .delta. 168.7, 161.3, 154.5, 153.3, 144.8,
140.1, 130.3, 120.3, 105.6, 96.6, 60.9, 56.2, 42.1, 28.5; IR (thin
film, cm.sup.-1) 2938, 1672, 1612, 1579, 1503, 1453, 1417, 1318,
1271, 1151, 1121, 1051, 1000, 824, 730; m/z found: 444.12
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.17H.sub.18INO.sub.5:
443.0230; found 443.0217.
##STR00075##
[0167] General procedure for Negishi Coupling:
[0168] Iodopiperlongumine (50.0 mg, 0.11 mmol) was added to a
solution of tris(dibenzylideneacetone)dipalladium(0)
(Pd.sub.2(dba).sub.3, 5.16 mg, 5.64 .mu.mol) and
2-dicyclohexylphosphino-2',6'-diisopropoxybiphenyl (RuPhos, 10.5
mg, 0.02 mmol) in dry dimethylacetamide (1.10 mL) at room
temperature. After the mixture had been stirred for 10 min,
alkylzinc bromide (0.23 mmol) was added immediately. After the
reaction mixture had been stirred at room temperature for 14 h, the
solvent was removed under reduced pressure. The residue was
purified by flash chromatography (hexane/ethyl acetate=100/0 to
60/40).
##STR00076##
(E)-3-methyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropyridi-
n-2(1H)-one
[0169] Yield: 99%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.65
(d, J=15.6 Hz, 1H), 7.39 (d, J=15.6 Hz, 1H), 6.79 (s, 2H), 6.69 (m,
1H), 4.00 (t, J=6.6 Hz, 2H), 3.89 (s, 6H), 3.86 (s, 3H), 2.41 (m,
2H), 1.94 (s, 3H) .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.1,
167.1, 153.3, 143.4, 140.1, 140.0, 131.9, 130.7, 121.3, 105.6,
60.9, 56.2, 42.3, 24.6, 16.9; IR (thin film, cm.sup.-1) 2938, 2839,
1677, 1652, 1615, 1580, 1504, 1453, 1418, 1388, 1352, 1318, 1293,
1276, 1242, 1204, 1154, 1125, 1092, 1050, 1004, 895, 853. m/z
found: 332.44 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.18H.sub.21NO.sub.5: 331.1420; found 331.1407.
##STR00077##
(E)-3-propyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropyridi-
n-2(1H)-one
[0170] Yield: 42%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.66
(d, J=15.6 Hz, 1H), 7.39 (d, J=15.3 Hz, 1H), 6.80 (s, 2H), 6.67 (t,
J=4.2 Hz, 1H), 3.99 (t, J=6.6 Hz, 2H), 3.89 (s, 6H), 3.88 (s, 3H),
2.43 (m, 2H), 2.31 (t, J=7.5 Hz, 2H), 1.47-1.54 (m, 2H), 0.95 (t,
J=7.5 Hz, 3H); .sup.13C NMR (126 MHz, CDCl.sub.3) .delta. 169.3,
166.7, 153.4, 143.4, 140.0, 139.5, 136.2, 130.8, 121.4, 105.6,
60.9, 56.2, 42.2, 32.5, 24.7, 21.8, 13.8; IR (thin film, cm.sup.-1)
2931, 2838, 1673, 1614, 1579, 1504, 1454, 1417, 1386, 1352, 1316,
1295, 1274, 1240, 1152, 1121, 1051, 1004, 975, 906, 844, 826; m/z
found: 360.60 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.20H.sub.25NO.sub.5: 359.1733; found 359.1733.
##STR00078##
(E)-3-cyclopropyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydrop-
yridin-2(1H)-one
[0171] Yield: 70%; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.66
(d, J=15.5 Hz, 1H), 7.46 (d, J=15.5 Hz, 1H), 6.78 (s, 2H), 6.43 (t,
J=4.5 Hz, 1H), 3.95 (t, J=6.5 Hz, 2H), 3.89 (s, 6H), 3.87 (s, 3H),
2.37-2.41 (m, 2H), 1.85 (m, 1H), 0.82 (dd, J=13.0, 5.0, 2H), 0.46
(dd, J=10.5, 5.0, 2H); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.
169.2, 167.0, 153.3, 143.5, 139.8, 137.5, 136.0, 130.7, 121.3,
105.5, 60.9, 56.2, 41.9, 24.3, 10.5, 6.6; IR (cm.sup.-1) 2937,
1672, 1613, 1579, 1503, 1464, 1417, 1383, 1348, 1316, 1275, 1243,
1152, 1123, 1052, 1003, 974, 879, 853, 827, 792, 750, 734, 700,
618.68; m/z found: 358.25 [M+H.sup.+].
##STR00079##
(E)-3-cyclohexyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropy-
ridin-2(1H)-one
[0172] Yield: 30%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.65
(d, J=15.6 Hz, 1H), 7.39 (d, J=15.6 Hz, 1H), 6.80 (s, 2H), 6.60 (t,
J=4.3 Hz, 1H), 3.95 (t, J=6.6 Hz, 2H), 3.89 (s, 6H), 3.87 (s, 3H),
2.63 (t, J=11.7 Hz, 1H), 2.42 (dd, J=11.1, 6.0, 2H), 1.61-1.81 (m,
5H), 1.04-1.45 (m, 6H); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.
169.7, 166.7, 153.5, 143.5, 141.7, 137.6, 133.6, 131.0, 121.8,
105.7, 61.2, 56.5, 42.2, 37.3, 33.1, 26.9, 26.5, 24.9; IR
(cm.sup.-1) 2925, 2850, 1673, 1613, 1580, 1504, 1450, 1417, 1388,
1351, 1316, 1295, 1240, 1274, 1179, 1124, 1151, 1096, 1052, 1003,
974, 952, 927, 905, 888, 854, 825, 780, 731, 701; m/z found: 400.34
[M+H.sup.+]; HRMS (FAB) calcd For C.sub.23H.sub.29NO.sub.5:
399.2046; found 399.2042.
##STR00080##
General procedure for Negishi Coupling:
[0173] A mixture of Cs.sub.2CO.sub.3 (74.0 mg, 0.23 mmol),
dichlorobis(tricyclohexylphosphine) palladium(II) (4.16 mg, 5.64
mmol), arylboronic acid (0.23 mmol) and compound iodopiperlongumine
(50.0 mg, 0.11 mmol) were stirred at reflux for 3 h or microwave at
100.degree. C. for 5 min in a 9:1 mixture of 1,4-dioxane and water
(1.20 ml). At the end, the solvent was evaporated under reduced
pressure and the residue was purified by flash column
chromatography (hexane/ethyl acetate=100/0 to 60/40) to give the
coupling products.
##STR00081##
(E)-3-phenyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropyridi-
n-2(1H)-one
[0174] Yield: 90%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.69
(d, J=15.3 Hz, 1H), 7.43 (d, J=15.3 Hz, 1H), 7.35-7.43 (m, 5H),
7.03 (t, J=4.2 Hz, 1H), 6.78 (s, 2H), 4.13 (t, J=6.3 Hz, 2H), 3.86
(s, 9H), 2.59-2.65 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 169.3, 165.8, 153.4, 143.9, 142.4, 140.1, 137.1, 136.0,
130.6, 128.7, 128.2, 128.1, 121.2, 105.7, 60.9, 56.3, 42.1, 25.1;
IR (thin film, cm.sup.-1) 2937, 2840, 1676, 1614, 1580, 1504, 1463,
1418, 1349, 1317, 1276, 1154, 1125, 1053, 1004, 826, 700; m/z
found: 394.18 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.23H.sub.23NO.sub.5: 393.1576; found 393.1568.
##STR00082##
(E)-3-(4-methoxyphenyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-di-
hydropyridin-2(1H)-one
[0175] Yield: 86%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.69
(d, J=15.6, 1H), 7.43 (d, J=15.3 Hz, 1H), 7.34 (d, J=8.7 Hz, 2H),
6.97 (t, J=4.5 Hz, 1H), 6.92 (d, J=8.7 Hz, 2H), 6.78 (s, 2H), 4.12
(t, J=6.3 Hz, 2H), 3.86 (s, 9H), 3.82 (s, 3H), 2.57-2.63 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.3, 166.0, 159.6,
153.3, 143.8, 141.2, 137.5, 136.5, 130.7, 129.9, 128.4, 121.2,
113.7, 105.7, 60.9, 56.2, 55.32, 42.1, 25.0; IR (cm.sup.-1) 2937,
2837, 1679, 1606, 1580, 1504, 1464, 1416, 1343, 1317, 1278, 1246,
1154, 1126, 1044, 1027, 1004, 879, 830, 802, 781, 734, 693, 598;
m/z found: 424.52 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.24H.sub.25NO.sub.6: 423.1682; found 423.1677.
##STR00083##
(E)-3-(4-fluorophenyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dih-
ydropyridin-2(1H)-one
[0176] Yield: 85%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.70
(d, J=15.6 Hz, 1H), 7.36-7.44 (m, 3H), 7.02-7.10 (m, 2H), 7.01 (d,
J=4.5 Hz, 1H), 6.78 (s, 2H), 4.12 (t, J=6.3 Hz, 2H), 3.86 (s, 9H),
2.59-2.65 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.3,
165.7, 162.2 (d, J=247.6 Hz, 1C), 153.3, 144.0, 142.4, 138.0 (d,
J=8.65 Hz, 1C), 136.1, 131.9 (d, J=3.38 Hz, 1C), 130.6, 130.4,
121.1, 115.1 (d, J=21.4 Hz, 1C), 105.7, 60.9, 56.2, 42.1, 25.0; IR
(thin film, cm.sup.-1) 2939, 2839, 1678, 1613, 1580, 1504, 1464,
1417, 1385, 1349, 1316, 1266, 1224, 1152, 1124, 1052, 1001, 930,
860, 880, 833, 812, 781, 730, 701; m/z found: 412.16 [M+H.sup.+];
HRMS (FAB) calcd for C.sub.23H.sub.22FNO.sub.5: 411.1482; found
411.1478.
##STR00084##
(E)-3-(4-(trifluoromethyl)phenyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-eno-
yl)-5,6-dihydropyridin-2(1H)-one
[0177] Yield: 99%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.71
(d, J=15.3 Hz, 1H), 7.65 (d, J=8.4 Hz, 1H), 7.53 (d, J=8.4 Hz, 1H),
7.41 (d, J=15.3 Hz, 1H), 7.09 (t, J=4.5 Hz, 1H), 6.78 (s, 2H), 4.15
(t, J=6. Hz, 2H), 3.86 (s, 9H), 2.63-2.69 (m, 2H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 169.2, 165.3, 153.4, 144.3, 143.6, 140.2,
139.5, 136.1, 130.5, 130.4, 127.9 (q, J=302.0 Hz, 1C), 129.2, 125.1
(q, J=3.6 Hz, 1C), 120.9, 105.7, 60.9, 56.2, 42.0, 25.1; IR (thin
film, cm.sup.-1) 2941, 1677, 1615, 1580, 1504, 1465, 1417, 1386,
1347, 1321, 1298, 1275, 1243, 1162, 1121, 1068, 1053, 1037, 1016,
1002, 934, 907, 882, 863, 837, 782; m/z found: 462.41 [M+H.sup.+];
HRMS (FAB) calcd for C.sub.24H.sub.22F.sub.3NO.sub.5: 461.1450;
found 461.1446.
##STR00085##
(E)-3-(4-(dimethylamino)phenyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl-
)-5,6-dihydropyridin-2(1H)-one
[0178] Yield: 50%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.68
(d, J=15.6 Hz, 1H), 7.44 (d, J=15.3 Hz, 1H), 7.30 (d, J=8.7 Hz,
2H), 6.94 (t, J=4.5 Hz, 1H), 6.78 (s, 2H), 6.73 (d, J=8.7 Hz, 2H),
4.11 (t, J=6.6 Hz, 2H), 3.87 (s, 9H), 2.97 (s, 6H), 2.55-2.61 (m,
2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.4, 166.2, 153.3,
150.4, 143.6, 140.0, 139.8, 136.7, 130.7, 129.4, 123.8, 121.4,
112.0, 105.6, 60.9, 56.2, 42.2, 40.4, 25.0; IR (thin film,
cm.sup.-1) 2937, 2838, 1672, 1609, 1579, 1521, 1503, 1452, 1417,
1383, 1347, 1316, 1266, 1226, 1199, 1150, 1122, 1051, 1002, 973,
946, 876, 853, 818, 781, 730, 700; m/z found: 437.50 [M+H.sup.+];
HRMS (FAB) calcd for C.sub.25H.sub.28N2O.sub.5: 436.1998; found
436.2000.
##STR00086##
(E)-3-(pyridin-3-yl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihyd-
ropyridin-2(1H)-one
[0179] Yield: 45%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.63
(d, J=1.5 Hz, 1H), 8.59 (dd, J=4.8, 1.5 Hz, 1H), 7.77-7.80 (m, 1H),
7.70 (d, J=15.6 Hz, 1H), 7.39 (d, J=15.6 Hz, 1H), 7.29-7.34 (m,
1H), 7.10 (t, J=4.5 Hz, 1H), 6.78 (s, 2H), 4.15 (t, J=6.3 Hz, 2H),
3.87 (s, 6H), 3.86 (s, 3H), 2.64-2.70 (m, 2H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 169.1, 165.3, 153.4, 149.3, 144.2, 143.6,
140.2, 136.4, 134.1, 131.8, 130.5, 122.8, 120.9, 105.7, 60.9, 56.2,
42.0, 25.1; IR (thin film, cm.sup.-1) 2940, 2839, 1674, 1614, 1580,
1504, 1464, 1417, 1386, 1350, 1317, 1266, 1242, 1154, 1123, 1055,
1001, 933, 881, 856, 826, 812, 789, 774, 729, 712, 700; m/z found:
395.14 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.22H.sub.22N.sub.2O.sub.5: 394.1529; found 394.1536.
##STR00087##
(E)-3-(benzo[b]thiophen-2-yl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)--
5,6-dihydropyridin-2(1H)-one
[0180] Yield: 53%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.62-7.75 (m, 4H), 7.38 (d, J=15.3 Hz, 1H), 7.25-7.28 (m, 3H), 6.76
(s, 2H), 4.04 (t, J=6.3 Hz, 2H), 3.83 (s, 6H), 3.81 (s, 3H),
2.57-2.62 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.2,
164.6, 153.4, 144.1, 141.9, 140.2, 139.8, 139.6, 137.1, 130.6,
130.2, 124.8, 124.4, 123.9, 123.8, 121.9, 121.1, 105.8, 60.9, 56.3,
41.7, 25.3; IR (thin film, cm.sup.-1) 2935, 1674, 1612, 1579, 1503,
1456, 1433, 1417, 1350, 1315, 1271, 1242, 1150, 1123, 1000, 904,
859, 825, 728, 700; m/z found: 450.34 [M+H.sup.+]; HRMS (FAB) calcd
for C.sub.25H.sub.23NO.sub.5S: 449.1297; found 449.1296.
##STR00088##
(E)-3-p-tolyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropyrid-
in-2(1H)-one
[0181] Yield: 98%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.69
(d, J=15.6 Hz, 1H), 7.43 (d, J=15.6 Hz, 1H), 7.29 (d, J=8.4 Hz,
2H), 7.20 (d, J=8.4 Hz, 2H), 7.00 (t, J=4.5 Hz, 1H), 6.78 (s, 2H),
4.13 (t, J=6.6 Hz, 2H), 3.87 (s, 9H), 2.58-2.63 (m, 2H), 2.37 (s,
3H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.3, 165.9, 153.3,
143.8, 141.7, 137.9, 137.0, 135.7, 133.1, 130.6, 128.9, 128.6,
121.2, 105.7, 60.9, 56.2, 42.1, 25.0, 21.2; IR (thin film,
cm.sup.-1) 2938, 2838, 1675, 1611, 1579, 1503, 1453, 1417, 1382,
1343, 1315, 1295, 1266, 1241, 1151, 1122, 1052, 1002, 973, 932,
906, 878, 857, 819, 776, 756, 731, 669; m/z found: 408.49
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.24H.sub.25NO.sub.5:
407.1733; found 407.1731.
##STR00089##
(E)-3-o-tolyl-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydropyrid-
in-2(1H)-one
[0182] Yield: 98%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.72
(d, J=15.6 Hz, 1H), 7.48 (d, J=15.5 Hz, 1H), 7.16-7.31 (m, 4H),
6.91 (t, J=4.2 Hz, 1H), 6.79 (s, 2H), 4.20 (t, J=6.3 Hz, 2H), 3.86
(s, 9H), 2.62-2.66 (m, 2H), 2.25 (s, 3H); NMR (75 MHz, CDCl.sub.3)
.delta. 169.1, 165.4, 153.3, 144.2, 143.2, 140.1, 137.9, 136.4,
136.07, 130.6, 130.0, 129.6, 128.4, 125.8, 121.0, 105.7, 60.9,
56.2, 42.1, 25.0, 20.0; IR (thin film, cm.sup.-1) 2939, 1677, 1613,
1580, 1504, 1384, 1454, 1417, 1341, 1316, 1274, 1242, 1152, 1124,
1101, 1053, 1002, 975, 930, 908, 881, 857, 826, 804, 788, 728, 701,
620; m/z found: 408.53 [M.sup.+]; HRMS (FAB) calcd for
C.sub.24H.sub.25NO.sub.5: 407.1733; found 407.1735
##STR00090##
(E)-3-(2-bromophenyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihy-
dropyridin-2(1H)-one
[0183] Yield: 61%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.63
(d, J=15.6 Hz, 1H), 7.55 (d, J=8.1 Hz, 1H), 7.41 (d, J=15.6 Hz,
1H), 7.28 (d, J=7.5 Hz, 1H), 7.14-7.22 (m, 2H), 6.84 (t, J=4.5 Hz,
1H), 6.70 (s, 2H), 4.13 (m, 2H), 3.79 (s, 9H), 2.53-2.59 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.9, 164.7, 153.3,
144.3, 143.6, 140.1, 138.0, 137.4, 132.6, 131.0, 130.6, 129.7,
127.5, 123.8, 121.0, 105.7, 60.9, 56.2, 41.9, 25.0; IR (thin film,
cm.sup.-1) 3055, 2938, 2838, 1675, 1613, 1580, 1503, 1464, 1417,
1384, 1346, 1316, 1272, 1242, 1153, 1124, 1052, 1021, 1002, 930,
880, 853, 826, 783, 755, 729, 700, 662, 634, 619, 606; m/z found:
472.41 [M+H.sup.+ (.sup.79Br)]; HRMS (FAB) calcd for
C.sub.23H.sub.22.sup.79BrNO.sub.5: 471.0681; found 471.0680.
##STR00091##
(E)-3-(2-oxo-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-1,2,5,6-tetrahydr-
opyridin-3-yl)benzoic acid
[0184] Yield: 30%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
8.02-8.07 (m, 2H), 7.60-7.66 (m, 2H), 7.43 (t, J=7.8 Hz, 1H), 7.34
(d, J=15.3 Hz, 1H), 7.04 (t, J=3.9 Hz, 1H), 6.72 (s, 2H), 4.09 (t,
J=6.6 Hz, 2H), 3.79 (s, 9H), 2.56-2.62 (m, 2H); m/z found: 437.91
[M+H.sup.+]; HRMS (FAB) calcd For C.sub.24H.sub.23NO.sub.7:
437.1475; found 437.1473.
##STR00092##
[0185] Yield: 77%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.94-7.97 (m, 2H), 7.72 (d, J=15.6 Hz, 1H), 7.56 (d, J=8.1 Hz, 1H),
7.32-7.49 (m, 6H), 6.76 (s, 2H), 4.26 (t, J=6.3 Hz, 2H), 3.85 (s,
3H), 3.83 (s, 6H), 2.72-2.78 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 169.2, 165.0, 156.0, 153.7, 153.3, 144.8,
144.0, 140.0, 132.4, 130.6, 128.1, 127.2, 124.5, 124.1, 122.8,
122.6, 121.1, 120.7, 120.5, 111.7, 105.6, 60.9, 56.2, 42.0, 25.2;
IR (thin film, cm.sup.-1) 3055, 2939, 1681, 1613, 1581, 1504, 1464,
1451, 1416, 1385, 1349, 1317, 1265, 1242, 1190, 1153, 1125, 1086,
1053; 1002, 936, 902, 867, 838, 827, 802, 776, 755, 729, 701, 624;
m/z found: 484.45 [M+H.sup.+]; HRMS (FAB) calcd For
C.sub.29H.sub.25NO.sub.6: 483.1682; found 483.1682.
##STR00093##
(E)-3-(4-phenoxyphenyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-di-
hydropyridin-2(1H)-one
[0186] Yield: 99%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.68
(d, J=15.3 Hz, 1H), 7.46 (d, J=15.6 Hz, 1H), 7.16-7.37 (m, 5H),
7.06 (t, J=7.5 Hz, 1H), 6.95-6.99 (m, 4H), 6.78 (s, 2H), 3.97 (t,
J=6.3 Hz, 2H), 3.89 (s, 3H), 3.88 (s, 6H), 2.49-2.55 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 169.0, 165.0, 157.4,
154.5, 153.3, 143.8, 143.3, 140.0, 134.5, 131.1, 130.7, 129.7,
129.6, 128.3, 123.6, 123.0, 121.2, 119.3, 118.4, 105.7, 60.9, 56.2,
41.8, 25.0; IR (thin film, cm.sup.-1) 2938, 1678, 1613, 1579, 1504,
1484, 1465, 1449, 1417, 1384, 1348, 1316, 1272, 1224, 1152, 1124,
1099, 1052, 1001, 930, 908, 886, 864, 847, 826, 801, 784, 753, 730,
693; m/z found: 486.54 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.29H.sub.27NO.sub.6: 485.1838; found 485.1838.
##STR00094##
[0187] General Procedure for Sonogashira Coupling: A flame dried
flask was charged with
bis-(triphenylphosphine)palladium(II)dichloride (15.8 mg, 23.0 mop,
iodopiperlongumine (100 mg, 0.23 mmol), 1,4-dioxane (2.20 mL) and
DIPEA (0.11 mL, 0.68 mmol) via syringe. After the resulting
solution was carefully degassed with nitrogen for 10 min, copper
iodide (6.45 mg, 34.0 .mu.mol) and one of the selected alkynes
(0.68 mmol) were added. The resulting solution was stirred for 2-12
h (using TLC and LC-MS to monitor the reaction process). Upon
completion, the reaction was diluted with saturated aqueous
NH.sub.4Cl, extracted with CH.sub.2Cl.sub.2 (3.times.5 mL) and
dried over MgSO.sub.4. The solution was filtered, concentrated and
then purified by flash chromatography (hexane/ethyl acetate=60/40
to 40/60) to afford the desired compound.
##STR00095##
(E)-3-(phenylethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihy-
dropyridin-2(1H)-one
[0188] Yield: 84%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.72
(d, J=15.6 Hz, 1H), 7.51-7.55 (m, 2H), 7.48 (d, J=15.6 Hz, 1H);
7.28-7.35 (m, 4H), 6.83 (s, 2H), 4.08 (t, J=6.3 Hz, 2H), 3.90 (s,
6H), 3.89 (s, 3H), 2.57-2.63 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.7, 163.6, 153.3, 148.7, 144.4, 140.1,
131.8, 130.5, 128.7, 128.3, 122.3, 121.4, 120.7, 105.7, 92.2, 83.4,
60.9, 56.2, 41.5, 25.2; IR (thin film, cm.sup.-1) 2937, 2839, 1678,
1614, 1580, 1503, 1464, 1418, 1388, 1350, 1318, 1274, 1243, 1168,
1125, 1085, 1050, 1003, 919, 903, 861, 826, 758, 734, 692, 670; m/z
found: 418.54 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.25H.sub.23NO.sub.5: 417.1576; found 417.1572.
##STR00096##
(E)-3-((4-methoxyphenyl)ethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoy-
l)-5,6-dihydropyridin-2(1H)-one
[0189] Yield: 54%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.62
(d, J=15.6 Hz, 1H), 7.40 (d, J=15.6 Hz, 1H), 7.39 (d, J=8.4 Hz,
2H), 7.18 (t, J=4.5 Hz, 1H), 6.78 (d, J=8.7 Hz, 2H), 6.74 (s, 2H),
3.99 (t, J=6.3 Hz, 2H), 3.82 (s, 6H), 3.80 (s, 3H), 3.73 (s, 3H),
2.48-2.54 (m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.8,
163.7, 159.9, 153.3, 147.9, 144.3, 140.1, 133.3, 130.5, 121.6,
120.8, 114.4, 113.9, 105.7, 92.3, 82.2, 60.9, 56.2, 55.2, 41.5,
25.2; IR (thin film, cm.sup.-1) 2935, 2838, 1675, 1601, 1580, 1504,
1463, 1417, 1388, 1349, 1317, 1273, 1246, 1166, 1123, 1083, 1049,
1028, 1001, 925, 902, 859, 828, 791, 773, 731, 700, 667; m/z found:
448.14 [M+H.sup.+]; HRMS (FAB) calcd for C.sub.26H.sub.25NO.sub.6:
447.1682; found 447.1681.
##STR00097##
(E)-3-((4-fluorophenyl)ethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl-
)-5,6-dihydropyridin-2(1H)-one
[0190] Yield: 82%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.70
(d, J=15.5 Hz, 1H), 7.44-7.51 (m, 3H), 7.29 (t, J=4.8 Hz, 1H), 7.03
(t, J=8.7 Hz, 2H), 6.82 (s, 2H), 4.07 (t, J=6.3 Hz, 2H), 3.90 (s,
6H), 3.88 (s, 3H), 2.57-2.63 (m, 2H); NMR (75 MHz, CDCl.sub.3)
.delta. 168.7, 163.5, 162.7 (d, J=248.9 Hz, 1C), 153.3, 148.8,
144.4, 140.1, 133.7 (d, J=8.4 Hz, 1C), 130.4, 121.3, 120.7, 118.4
(d, J=3.53 Hz, 1C), 115.6 (d, J=22.05 Hz, 1C), 105.7, 91.1, 83.1,
60.8, 56.2, 41.5, 25.2; IR (thin film, cm.sup.-1) 2925, 1677, 1614,
1597, 1580, 1504, 1464, 1417, 1389, 1349, 1317, 1272, 1227, 1155,
1167, 1124, 1050, 1001, 902, 861, 835, 797, 773, 731, 701, 667,
620; m/z found: 436.15 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.25H.sub.22FNO.sub.5: 435.1482; found 435.1477.
##STR00098##
(E)-3((4-(trifluoromethoxy)phenyl)ethynyl)-1-(3-(3,4,5-trimethoxyphenyl)p-
rop-2-enoyl)-5,6-dihydropyridin-2(1H)-one
[0191] Yield: 63%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.70
(d, J=15.6 Hz, 1H), 7.54 (d, J=8.7 Hz, 2H), 7.45 (d, J=15.6 Hz,
1H), 7.30 (t, J=4.5 Hz, 1H), 7.17 (d, J=8.4 Hz, 2H), 6.81 (s, 2H),
4.07 (t, J=6.3 Hz, 2H), 3.88 (s, 6H), 3.87 (s, 3H), 2.57-2.63 (m,
2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.7, 163.5, 153.3,
149.2, 144.6, 140.2, 133.3, 130.4, 121.3, 121.2, 120.7, 120.6,
120.3 (q, J=256.4 Hz, 1C), 114.6, 105.7, 90.7, 84.2, 60.9, 56.2,
41.5, 25.3; IR (cm.sup.-1) 2924, 2852, 1678, 1614, 1580, 1504,
1464, 1418, 1389, 1350, 1318, 1250, 1204, 1157, 1122, 1083, 1050,
1002, 920, 902, 854, 825, 799, 774, 734, 703, 669; m/z found:
502.14 [M+H.sup.+]; HRMS (FAB) calcd For
C.sub.26H.sub.22F.sub.3NO.sub.6: 501.1399; found 501.1398.
##STR00099##
(E)-3-(cyclohexenylethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,-
6-dihydropyridin-2(1H)-one
[0192] Yield: 76%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.65
(d, J=15.6 Hz, 1H), 7.43 (d, J=15.6 Hz, 1H), 7.13 (t, J=4.5 Hz,
1H), 6.78 (s, 2H), 6.21 (s, 1H), 4.01 (t, J=6.6 Hz, 2H), 3.87 (s,
6H), 3.85 (s, 3H), 2.49-2.55 (m, 2H), 2.08-2.16 (m, 4H), 1.50-1.66
(m, 4H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.8, 163.7,
153.3, 147.6, 144.2, 140.0, 136.5, 130.5, 121.6, 120.8, 120.0,
105.6, 94.2, 80.8, 60.9, 56.2, 41.5, 28.9, 25.7, 25.1, 22.1, 21.3;
IR (thin film, cm.sup.-1) 2923, 2853, 1674, 1613, 1580, 1504, 1454,
1417, 1388, 1348, 1317, 1273, 1242, 1153, 1123, 1080, 1050, 1002,
974, 917, 901, 860, 825, 799, 774, 731, 701, 670; m/z found: 422.19
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.25H.sub.27NO.sub.5:
421.1889; found 421.1890.
##STR00100##
(E)-3-((2-chlorophenyl)ethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl-
)-5,6-dihydropyridin-2(1H)-one
[0193] Yield: 68%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.63
(d, J=15.3 Hz, 1H), 7.49 (dd, J=7.2, 2.1, 1H), 7.39 (d, J=15.6,
1H), 7.28-7.37 (m, 2H), 7.13-7.22 (m, 2H), 6.74 (s, 2H), 4.01 (t,
J=6.3 Hz, 2H), 3.82 (s, 6H), 3.80 (s, 3H), 2.51-2.57 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.7, 163.4, 153.3,
149.6, 144.5, 140.1, 136.0, 133.5, 130.5, 129.7, 129.2, 126.4,
122.4, 121.3, 120.7, 105.7, 88.9, 88.4, 60.9, 56.2, 41.5, 25.3; IR
(thin film, cm.sup.-1) 2924, 2852, 1674, 1613, 1579, 1503, 1464,
1417, 1388, 1349, 1316, 1291, 1272, 1242, 1167, 1146, 1122, 1086,
1053, 1001, 974, 955, 925, 902, 861, 825, 755, 732, 701, 680; m/z
found: 452.13 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.25H.sub.22ClNO.sub.5: 451.1187; found 451.1187.
##STR00101##
(E)-3-((2-fluorophenyl)ethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl-
)-5,6-dihydropyridin-2(1H)-one
[0194] Yield: 87%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.69
(d, J=15.6, 1H), 7.48-7.53 (m, 1H), 7.46 (d, J=15.3, 1H), 7.26-7.34
(m, 2H), 7.05-7.12 (m, 2H), 6.80 (s, 2H), 4.07 (t, J=6.6 Hz, 2H),
3.88 (s, 6H), 3.86 (s, 3H), 2.57-2.63 (m, 2H); .sup.13C NMR (75
MHz, CDCl.sub.3) .delta. 168.7, 163.4, 162.7 (d, J=250.65 Hz, 1C),
153.3, 149.5, 144.5, 140.2, 133.6, 130.6, 130.5, 123.9 (d, J=3.68
Hz, 1C), 121.3, 120.7, 115.5 (d, J=20.70 Hz, 1C), 111.1 (d, J=15.6
Hz, 1C), 105.7, 88.3 (d, J=3.23 Hz, 1C), 85.5, 60.9, 56.2, 41.5,
25.3; IR (thin film, cm.sup.-1) 2924, 2852, 1679, 1614, 1580, 1504,
1491, 1451, 1417, 1387, 1349, 1317, 1273, 1242, 1168, 1154, 1124,
1103, 1081, 1050, 1001, 974, 925, 902, 862, 828, 797, 758, 731,
701; m/z found: 435.15 [M.sup.+]; HRMS (FAB) calcd for
C.sub.25H.sub.22FNO.sub.5: 435.1482; found 435.1483.
##STR00102##
(E)-3-(cyclopropylethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-
-dihydropyridin-2(1H)-one
[0195] Yield: 68%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.65
(d, J=15.6 Hz, 1H), 7.42 (d, J=15.6, 1H), 7.09 (t, J=4.5 Hz, 1H),
6.78 (s, 2H), 3.99 (d, J=6.3 Hz, 2H), 3.87 (s, 6H), 3.85 (s, 3H),
2.46-2.52 (m, 2H), 1.36-1.47 (m, 1H), 0.75-0.88 (m, 4H); .sup.13C
NMR (75 MHz, CDCl.sub.3) .delta. 168.8, 164.0, 153.3, 147.6, 144.2,
140.1, 130.5, 121.5, 120.8, 105.6, 96.7, 69.9, 60.9, 56.2, 41.5,
25.0, 8.7, 0.1; IR (thin film, cm.sup.-1) 2939, 1684, 1615, 1581,
1504, 1464, 1418, 1389, 1319, 1276, 1246, 1166, 1155, 1125, 1083,
1051, 1003, 931, 892, 858, 826, 775, 734, 621; m/z found: 382.42
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.22H.sub.23NO.sub.5:
381.1576; found 381.1570.
##STR00103##
(E)-3-(cyclopentylethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-
-dihydropyridin-2(1H)-one
[0196] Yield: 76%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.65
(d, J=15.6 Hz, 1H), 7.43 (d, J=15.3 Hz, 1H), 7.10 (t, J=4.5 Hz,
1H), 6.78 (s, 2H), 3.99 (t, J=6.3 Hz, 2H), 3.87 (s, 6H), 3.85 (s,
3H), 2.80 (p, J=7.5 Hz, 1H), 2.46-2.53 (m, 2H), 1.95-2.02 (m, 2H),
1.54-1.70 (m, 6H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.8,
164.0, 153.2, 147.4, 144.2, 140.0, 130.5, 121.6, 120.9, 105.6,
97.9, 74.2, 60.9, 56.2, 41.6, 33.7, 30.6, 25.0; IR (thin film,
cm.sup.-1) 2923, 2869, 1674, 1614, 1579, 1504, 1453, 1417, 1386,
1316, 1273, 1244, 1152, 1122, 1051, 1002, 973, 932, 897, 857, 824,
775, 732, 701, 669; m/z found: 410.45 [M+H.sup.+]; HRMS (FAB) calcd
for C.sub.24H.sub.27NO.sub.5: 409.1899; found 409.1883.
##STR00104##
(E)-3-(cyclohexylethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6--
dihydropyridin-2(1H)-one
[0197] Yield: 70%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.65
(d, J=15.6 Hz, 1H), 7.42 (d, J=15.6 Hz, 1H), 7.11 (t, J=4.5 Hz,
1H), 6.78 (s, 2H), 4.00 (t, J=6.3 Hz, 2H), 3.87 (s, 6H), 3.85 (s,
3H), 2.46-2.57 (m, 3H), 1.28-1.90 (m, 10H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.8, 164.0, 153.3, 147.5, 144.1, 140.0,
130.5, 121.6, 120.9, 105.6, 97.6, 74.7, 60.8, 56.2, 41.6, 32.4,
29.6, 25.7, 25.0, 24.8; IR (thin film, cm.sup.-1) 2928, 2852, 1677,
1614, 1580, 1504, 1450, 1417, 1386, 1348, 1317, 1274, 1153, 1124,
1052, 1003, 976, 931, 889, 857, 825, 776, 733, 700; m/z found:
424.51 [M+H.sup.+]; HRMS (FAB) calcd for C.sub.25H.sub.29NO.sub.5:
423.2046; found 423.2049.
##STR00105##
(E)-3-(3,3-dimethylbut-1-ynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-
-5,6-dihydropyridin-2(1H)-one
[0198] Yield: 88%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.63
(d, J=15.6 Hz, 1H), 7.40 (d, J=15.6 Hz, 1H), 7.09 (t, J=4.5 Hz,
1H), 6.77 (s, 2H), 3.97 (t, J=6.3 Hz, 2H), 3.86 (s, 6H), 3.83 (s,
3H), 2.44-2.50 (m, 2H), 1.26 (s, 9H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.8, 163.8, 153.2, 147.5, 144.0, 140.0,
130.5, 121.4, 120.9, 105.6, 101.5, 73.2, 60.8, 56.1, 41.6, 30.7,
27.9, 25.0; IR (thin film, cm.sup.-1) 2968, 1673, 1614, 1580, 1504,
1454, 1417, 1386, 1345, 1317, 1273, 1241, 1152, 1123, 1092, 1051,
1002, 976, 928, 904, 868, 849, 825, 795, 774, 732, 701, 666; m/z
found: 398.46 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.23H.sub.27NO.sub.5: 397.1889; found 397.1882.
##STR00106##
(E)-3-(hex-1-ynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihydro-
pyridin-2(1H)-one
[0199] Yield: 59%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.66
(d, J=15.6 Hz, 1H), 7.43 (d, J=15.6 Hz, 1H), 7.11 (t, J=4.5 Hz,
1H), 6.78 (s, 2H), 4.00 (t, J=6.3, 2H), 3.87 (s, 6H), 3.86 (s, 3H),
2.47-2.53 (m, 2H), 2.39 (t, J=6.9 Hz, 2H), 1.38-1.60 (m, 4H), 0.90
(t, J=7.2 Hz, 3H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.8,
164.1, 153.3, 147.6, 144.2, 140.1, 130.5, 121.6, 120.8, 105.6,
93.8, 74.7, 60.9, 56.2, 41.5, 30.5, 25.0, 22.0, 19.1, 13.5; IR
(cm.sup.-1) 2939, 2843, 1668, 1592, 1511, 1452, 1423, 1383, 1341,
1318, 1297, 1244, 1211, 1157, 1182, 1140, 1109, 1053, 1038, 989,
913, 862, 820, 801, 734, 700, 673, 636, 594; m/z found: 398.47
[M+H.sup.+]; HRMS (FAB) calcd for C.sub.23H.sub.27NO.sub.5:
397.1889; found 397.1882.
##STR00107##
(E)-3-(3-(dimethylamino)prop-1-ynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2--
enoyl)-5,6-dihydropyridin-2(1H)-one
[0200] Yield: 42%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.67
(d, J=15.6 Hz, 1H), 7.41 (d, J=15.3 Hz, 1H), 7.20 (t, J=4.5 Hz,
1H), 6.79 (s, 2H), 4.03 (t, J=6.6 Hz, 2H), 3.88 (s, 6H), 3.86 (s,
3H), 3.45 (s, 2H), 2.51-2.56 (m, 2H), 2.33 (s, 6H); .sup.13C NMR
(75 MHz, CDCl.sub.3) .delta. 168.7, 163.8, 153.3, 148.6, 144.4,
140.1, 130.5, 121.2, 120.7, 105.7, 88.1, 79.4, 60.9, 56.2, 48.5,
44.2, 41.5, 25.1; IR (thin film, cm.sup.-1) 2940, 1675, 1614, 1580,
1504, 1454, 1417, 1387, 1350, 1316, 1272, 1243, 1152, 1124, 1050,
1001, 929, 826, 775, 729, 700; m/z found: 399.16 [M+H.sup.+];
Compound decomposes slowly at room temperature.
##STR00108##
(E)-3-(dodec-1-ynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,6-dihyd-
ropyridin-2(1H)-one
[0201] Yield: 58%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.65
(d, J=15.3 Hz, 1H), 7.43 (d, J=15.6 Hz, 1H), 7.11 (t, J=4.5 Hz,
1H), 6.78 (s, 2H), 4.00 (t, J=6.3 Hz, 2H), 3.87 (s, 6H), 3.85 (s,
3H), 2.47-2.53 (m, 2H), 2.37 (t, J=7.2 Hz, 2H), 1.51-1.60 (m, 2H),
1.23-1.38 (s, 14H), 0.84 (t, J=6.9 Hz, 3H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.7, 164.0, 153.3, 147.6, 144.2, 140.0,
130.5, 121.6, 120.8, 105.6, 93.8, 74.6, 60.8, 56.2, 41.5, 31.8,
29.5, 29.4, 29.2, 29.0, 28.9, 28.5, 25.0, 22.6, 19.4, 14.0; IR
(thin film, cm.sup.-1) 2924, 2853, 1675, 1615, 1580, 1504, 1464,
1417, 1386, 1350, 1317, 1274, 1243, 1225, 1153, 1124, 1100, 1050,
1004, 977, 825, 775, 733, 702, 620, 608; m/z found: 482.62
[M.sup.+]; HRMS (FAB) calcd for C.sub.29H.sub.39NO.sub.5: 481.2828;
found 481.2827.
##STR00109##
(E)-3-(pyridin-3-ylethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,-
6-dihydropyridin-2(1H)-one
[0202] Yield: 82%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 8.76
(s, 1H), 8.56 (d, J=4.5 Hz, 1H), 7.82 (d, J=7.8 Hz, 1H), 7.72 (d,
J=15.6 Hz, 1H), 7.47 (d, J=15.3 Hz, 1H), 7.36 (t, J=4.5 Hz, 1H),
7.29 (t, J=4.8 Hz, 1H), 6.82 (s, 2H), 4.09 (t, J=6.3 Hz, 2H), 3.90
(s, 6H), 3.88 (s, 3H), 2.61-2.66 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.7, 163.3, 153.3, 152.3, 149.7, 149.0,
144.6, 140.2, 138.6, 130.4, 122.9, 121.1, 120.6, 119.6, 105.7,
88.7, 86.6, 60.9, 56.2, 41.4, 25.3; IR (thin film, cm.sup.-1) 2939,
2839, 1681, 1615, 1581, 1504, 1465, 1418, 1351, 1319, 1275, 1170,
1125, 1022, 1003, 826, 774, 705; HRMS (FAB) calcd for
C.sub.24H.sub.22N.sub.2O.sub.5: 418.1529; found 418.1533.
##STR00110##
(E)-3-((3-chlorophenyl)ethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl-
)-5,6-dihydropyridin-2(1H)-one
[0203] Yield: 81%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.71
(d, J=15.3 Hz, 1H), 7.39-7.52 (m, 3H), 7.24-7.33 (m, 3H), 6.82 (s,
2H), 4.08 (t, J=6.6 Hz, 2H), 3.90 (s, 6H), 3.88 (s, 3H), 2.59-2.64
(m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.7, 163.4,
153.3, 149.3, 144.6, 140.2, 134.2, 131.6, 130.4, 129.9, 129.5,
129.0, 124.1, 121.2, 120.6, 105.7, 90.7, 84.6 60.9, 56.2, 41.5,
25.3; IR (thin film, cm.sup.-1) 2936, 1679, 1615, 1581, 1504, 1465,
1418, 1388, 1350, 1319, 1275, 1243, 1170, 1147, 1126, 1003, 826,
787; m/z found: 452.13 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.25H.sub.22ClNO.sub.5: 451.1187; found 451.1182.
##STR00111##
(E)-3-(thiophen-3-ylethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5-
,6-dihydropyridin-2(1H)-one
[0204] Yield: 70%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.71
(d, J=15.3 Hz, 1H), 7.56 (d, J=2.1 Hz, 1H), 7.48 (d, J=15.6 Hz,
1H), 7.28-7.30 (m, 2H), 7.19 (d, J=5.1 Hz, 1H), 6.82 (s, 2H), 4.08
(t, J=6.3 Hz, 2H), 3.90 (s, 6H), 3.88 (s, 3H), 2.57-2.62 (m, 2H);
.sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.8, 163.6, 153.3,
148.6, 144.5, 140.2, 130.5, 129.8, 129.6, 125.4, 121.5, 121.4,
120.7, 105.7, 87.4, 83.0, 60.9, 56.2, 41.5, 25.3; IR (thin film,
cm.sup.-1) 3104, 2935, 2829, 1673, 1613, 1579, 1503, 1463, 1417,
1385, 1350, 1316, 1274, 1242, 1153, 1121, 1049, 1000, 971, 857,
824, 785; HRMS (FAB) calcd for C.sub.23H.sub.21NO.sub.5S: 423.1140;
found 423.1137.
##STR00112##
(E)-3-(3-phenylprop-1-ynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2-enoyl)-5,-
6-dihydropyridin-2(1H)-one
[0205] Yield: 43%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.70
(d, J=15.6 Hz, 1H), 7.46 (d, J=15.6 Hz, 1H), 7.24-7.40 (m, 5H),
7.20 (d, J=4.8 Hz, 1H), 6.81 (s, 2H), 4.04 (t, J=6.3 Hz, 2H), 3.89
(s, 6H), 3.88 (s, 3H), 3.83 (s, 2H), 2.51-2.56 (m, 2H); .sup.13C
NMR (75 MHz, CDCl.sub.3) .delta. 168.8, 164.0, 153.3, 148.3, 144.4,
140.1, 136.1, 130.5, 128.5, 127.9, 126.7, 121.4, 120.8, 105.7,
90.9, 76.8, 60.9, 56.2, 41.5, 25.7, 25.1; HRMS (FAB) calcd for
C.sub.26H.sub.25NO.sub.5: 431.1733; found 431.1733.
##STR00113##
(E)-3-((1-hydroxycyclohexyl)ethynyl)-1-(3-(3,4,5-trimethoxyphenyl)prop-2--
enoyl)-5,6-dihydropyridin-2(1H)-one
[0206] Yield: 95%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.64
(d, J=15.6 Hz, 1H), 7.36 (d, J=15.3 Hz, 1H), 7.15 (d, J=4.5 Hz,
1H), 6.78 (s, 2H), 3.98 (t, J=6.3 Hz, 2H), 3.87 (s, 6H), 3.85 (s,
3H), 3.04 (br, 1H), 2.47-2.53 (m, 2H), 1.86-1.97 (m, 2H), 1.50-1.69
(m, 8H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.8, 163.6,
153.3, 148.6, 144.2, 140.1, 130.4, 120.9, 120.8, 105.7, 96.2, 78.2,
68.7, 56.2, 41.5, 39.7, 25.1, 25.06, 23.1; HRMS (FAB) calcd for
C.sub.25H.sub.29NO.sub.6: 439.1995; found 439.1994.
1-Methacryloyl-5,6-dihydropyridin-2(1H)-one
[0207] .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. 2.01 (t, J=1.2
Hz, 3H), 2.50 (tdd, J=6.4, 6.4, 4.3, 1.9 Hz, 2H), 3.89 (t, J=6.5
Hz, 2H), 5.24 (quin, J=1.4 Hz, 1H), 5.29 (t, J=0.9 Hz, 1H), 5.98
(dt, J=9.7, 1.9 Hz, 1H), 6.93 (dt, J=9.7, 4.1 Hz, 1H); .sup.13C NMR
(75 MHz, CHLOROFORM-d) .delta. 18.9, 24.7, 42.3, 117.6, 125.2,
142.6, 145.3, 165.1, 175.0. Exact mass (M+H).sup.+ calc'd:
166.0868; found: 166.0862.
##STR00114##
1,4-bis(2-Oxo-5,6-dihydropyridin-1(2H)-yl)butane-1,4-dione
[0208] .sup.1H NMR (500 MHz, CHLOROFORM-d) .delta..quadrature.ppm
2.41 (dtd, J=8.5, 4.3, 4.3, 2.0 Hz, 4H), 3.26 (s, 4H), 3.95 (t,
J=6.6 Hz, 4H), 5.98 (dt, J=9.8, 2.0 Hz, 2H), 6.76-6.95 (m, 2H);
.sup.13C NMR (126 MHz, CHLOROFORM-d) .delta. 24.5, 34.3, 40.9,
125.8, 145.1, 165.2, 175.3. Exact mass (M+Na).sup.+ calc'd:
299.1008; found 299.1013.
##STR00115##
1,1'-((2E,2'E)-3,3'-(1,4-phenylene)bis(acryloyl)bis(5,6-dihydropyridin-2(-
1H)-one)
[0209] .sup.1H NMR (500 MHz, CHLOROFORM-d) .delta. 2.49 (tdd,
J=6.5, 6.5, 4.4, 1.7 Hz, 4H), 4.05 (t, J=6.3 Hz, 4H), 6.06 (dt,
J=9.6, 1.8 Hz, 2H), 6.95 (dt, J=9.3, 4.4 Hz, 2H), 7.54 (d, J=15.6
Hz, 1H), 7.59 (s, 3H), 7.73 (d, J=15.6 Hz, 2H); .sup.13C NMR (126
MHz, CHLOROFORM-d) .delta..quadrature.24.8, 41.6, 122.7, 125.8,
128.7, 136.6, 142.5, 145.5, 165.7, 168.8. Exact mass (M+Na).sup.+
calc'd: 399.1321; found 399.1319.
##STR00116##
1-Acryloyl-5,6-dihydropyridin-2(1H)-one
[0210] .sup.1H NMR (500 MHz, CHLOROFORM-d) .delta. 2.47 (tdd,
J=6.3, 6.3, 4.4, 2.0 Hz, 2H), 4.00 (t, J=6.6 Hz, 2H), 5.76 (dd,
J=10.3, 1.5 Hz, 1H), 6.02 (dt, J=9.8, 1.7 Hz, 1H), 6.40 (dd,
J=17.1, 2.0 Hz, 1H), 6.87-6.98 (m, 1H), 7.06 (dd, J=16.8, 10.5 Hz,
1H); .sup.13C NMR (126 MHz, CHLOROFORM-d) .delta. 24.7, 41.4,
125.6, 128.5, 131.6, 145.7, 165.5, 168.8. Exact mass (M+H).sup.+
calc'd: 152.0712: found: 152.0699
##STR00117##
1-(3-Phenylpropioloyl)-5,6-dihydropyridin-2(1H)-one
[0211] .sup.1H NMR (500 MHz, CHLOROFORM-d) .delta. 2.43-2.53 (m,
2H), 4.06 (t, J=6.3 Hz, 2H), 6.07 (dt, J=9.8, 1.5 Hz, 1H),
6.93-6.99 (m, 1H), 7.33-7.40 (m, 2H), 7.41-7.46 (m, 2H), 7.65 (d,
J=7.8 Hz, 1H); .sup.13C NMR (126 MHz, CHLOROFORM-d) .delta. 24.4,
40.8, 83.7, 93.3, 120.5, 125.1, 128.4, 130.4, 133.0, 145.6, 153.0,
163.7. Exact mass (M+Na).sup.+ calc'd: 248.0687; found
248.0685.
##STR00118##
(E)-1,4-bis(2-oxo-5,6-dihydropyridin-1(2H)-yl)but-2-ene-1,4-dione
[0212] .sup.1H NMR (500 MHz, CHLOROFORM-d+few drops of METHANOL-d4)
.delta. 7.56 (s, 2H), 6.97-6.87 (m, 2H), 5.96 (d, J=9.8 Hz, 2H),
3.94 (t, J=6.5 Hz, 4H), 2.48-2.38 (m, 4H); .sup.13C NMR (126 MHz,
CHLOROFORM-d) .delta. 168.1, 165.6, 146.6, 134.1, 125.1, 41.6,
24.7. Exact mass (M+H).sup.+ calc'd: 275.1032, found 275.1039
##STR00119##
(E)-N-methacryloylbut-2-enamide
[0213] .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. 8.35 (s, 1H),
7.25-6.90 (m, 2H), 5.79 (d, J=0.6 Hz, 1H), 5.56 (dd, J=3.0, 1.4 Hz,
1H), 1.94 (s, 3H), 1.89 (dd, J=6.5, 1.2 Hz, 3H); .sup.13C NMR (75
MHz, CHLOROFORM-d) .delta. 167.4, 166.8, 147.1, 140.1, 124.3,
122.7, 18.7, 18.6. Exact mass (M+Na).sup.+ calc'd: 176.0687, found
176.0690
##STR00120##
[0214] Experimental Procedure:
[0215] Piperlongumine (300 mg, 0.94 mmol) was dissolved in
CH.sub.2Cl.sub.2 (6 mL). Aluminum trichloride (874 mg, 6.55 mmol)
was added portion-wise at 0.degree. C. The reaction was then warmed
to room temperature and stirred for 1 h. At the end, the reaction
was quenched with saturated aqueous NH.sub.4Cl solution (1 mL): the
aqueous layer was extracted twice with CH.sub.2Cl.sub.2(3 mL) and
the organic layers were collected, washed with saturated aqueous
NaCl solution (3 mL) and dried over MgSO.sub.4. The solution was
filtrated and the solvent was evaporated under vacuum. The crude
was subjected to flash chromatography (hexane/ethyl acetate=100/0
to 60/40) to afford the monodemethylated product in 80% yield.
Note: It's very difficult to separate the desired product from the
bis-demethylated byproduct. But the quality of the desired product
is very important for the next Mitsunobu reaction.
[0216] Yield: 80%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.63
(d, J=15.3 Hz, 1H), 7.33 (d, J=15.6 Hz, 1H), 6.85-6.91 (m, 1H),
6.76 (s, 2H), 6.06 (s, 1H), 5.99 (d, J=9.6 Hz, 1H), 3.97 (t, J=6.6
Hz, 2H), 3.84 (s, 6H), 2.38-2.43 (m, 2H); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. 168.8, 165.7, 147.0, 145.4, 144.1, 137.0,
126.3, 125.6, 119.4, 105.2, 56.1, 41.5, 24.6; IR (thin film,
cm.sup.-1) 3368, 2939, 1676, 1593, 1511, 1455, 1424, 1384, 1344,
1316, 1286, 1243, 1212, 1181, 1157, 1136, 1110, 1053, 975, 912,
819, 798, 734, 701, 674; m/z found: 304.15 [M+H.sup.+]; HRMS (FAB)
calcd for C.sub.16H.sub.17NO.sub.5: 303.1107; found 303.1111.
##STR00121##
[0217] Experimental Procedure:
[0218] Triphenylphosphine (92.0 mg, 0.35 mmol) and (E)-diisopropyl
diazene-1,2-dicarboxylate (DIAD, 71.0 mg, 0.35 mmol) were dissolved
to a 1:1 mixture of THF and toluene (4 mL); then mono-demethylated
piperlongumine (50.0 mg, 0.18 mmol) was added. After 10 min,
2-(dimethylamino) ethanol (31.0 mg, 0.35 mmol) was added to the
mixture. The reaction was stirred at room temperature for 3 h. At
the end, the solvent was evaporated under vacuum, and the crude
product was subjected to flash chromatography (hexane/ethyl
acetate=100/0 to 50:50), to afford the desired product in 90%
yield.
[0219] Yield: 90%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.67
(d, J=15.6 Hz, 1H), 7.41 (d, J=15.6 Hz, 1H), 6.91-6.97 (m, 1H),
6.78 (s, 2H), 6.04 (d, J=9.9 Hz, 1H), 4.10 (t, J=5.7 Hz, 2H), 4.03
(t, J=6.3 Hz, 2H), 3.86 (s, 6H), 2.76 (t, J=6.0 Hz, 2H), 2.44-2.50
(m, 2H), 2.39 (s, 6H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
168.8, 165.8, 153.5, 145.4, 143.8, 139.0, 130.7, 125.9, 121.1,
105.5, 70.7, 58.7, 56.1, 45.5, 41.6, 24.8; IR (thin film,
cm.sup.-1) 2935, 1683, 1614, 1580, 1502, 1463, 1418, 1386, 1352,
1317, 1276, 1243, 1215, 1182, 1125, 1156, 1052, 1033, 999, 974,
912, 825, 731, 700, 634, 593; m/z found: 374.98 [M+H.sup.+]; HRMS
(FAB) calcd for C.sub.20H.sub.26N.sub.2O.sub.5: 374.1842; found
374.1845.
##STR00122##
[0220] Experimental Procedure:
[0221] Triphenylphosphine (77.0 mg, 0.29 mmol) and DIAD (60.0 mg,
0.29 mmol) were dissolved to a 1:1 mixture of THF and toluene (2
mL); then mono-demethylated piperlongumine (40.0 mg, 0.13 mmol) was
added. After 10 min, N-methyldiethanolamine (120 mg, 0.06 mmol) was
added to the mixture; the reaction was stirred at room temperature
for 3 h. At the end, the solvent was evaporated under vacuum. The
crude product was purified by preparative
TLC(CH.sub.2Cl.sub.2/MeOH=9/1) to afford the desired product in 78%
yield.
[0222] Yield: 78%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7:66
(d, J=15.6 Hz, 2H), 7.40 (d, J=15.3 Hz, 2H), 6.91-6.96 (m, 2H),
6.77 (s, 4H), 6.03 (d, J=9.9 Hz, 2H), 4.14 (t, J=6.0 Hz, 4H), 4.03
(t, J=6.3 Hz, 4H), 3.84 (s, 12H), 2.98 (m, 4H), 2.52 (s, 3H),
2.44-2.48 (m, 4H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.8,
165.8, 153.5, 145.4, 143.8, 139.1, 130.6, 125.8, 121.0, 105.5,
70.7, 57.0, 56.1, 42.8, 41.6, 24.8; IR (thin film, cm.sup.-1) 2936,
1684, 1614, 1581, 1502, 1464, 1418, 1386, 1352, 1317, 1265, 1243,
1215, 1183, 1156, 1127, 1053, 1038, 998, 912, 826, 730, 701, 594;
m/z found: 690.29 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.37H.sub.43N.sub.3O.sub.10: 689.2948; found 689.2949.
##STR00123##
[0223] Experimental Procedure:
[0224] Triphenylphosphine (79.0 mg, 0.30 mmol) and DIAD (61.0 mg,
0.30 mmol) were dissolved to a 1:1 mixture of THF and toluene (2
mL); then mono-demethylated piperlongumine (46.0 mg, 0.15 mmol) was
added. After 10 min, triethanolamine (6.80 mg, 0.05 mmol) was added
to the mixture. The reaction was stirred at room temperature for 3
h. At the end, the solvent was evaporated under vacuum. The crude
product was purified by preparative TLC(CH.sub.2Cl.sub.2/MeOH=9/1)
to afford the desired product in 47% yield.
[0225] Yield: 47%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.66
(d, J=15.6 Hz, 3H), 7.40 (d, J=15.3 Hz, 3H), 6.91-6.97 (m, 3H),
6.76 (s, 6H), 6.04 (d, J=9.6 Hz, 3H), 4.10 (m, 6H), 4.03 (t, J=6.3
Hz, 6H), 3.81 (s, 18H), 3.10 (m, 6H), 2.45-2.49 (m, 6H); .sup.13C
NMR (75 MHz, CDCl.sub.3) .delta. 168.9, 165.8, 153.5, 145.3, 143.9,
139.4, 130.4, 125.9, 120.9, 105.6, 71.5, 56.1, 54.6, 41.6, 24.8; IR
(cm.sup.-1) 2925, 2853, 1684, 1614, 1580, 1502, 1464, 1418, 1386,
1352, 1317, 1243, 1275, 1215, 1156, 1182, 1127, 1053, 1038, 997,
912, 826, 731; m/z found: 1005.41 [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.54H.sub.60N.sub.4O.sub.15: 1004.4055; found 1004.4050.
##STR00124##
3-methylene-5-[(E)-2-(3,4,5-trimethoxyphenyl)vinyl]dihydrofuran-2(3H)-one
[0226] Experimental Procedure:
[0227] To a mixture of (2E)-3-(3,4,5-trimethoxyphenyl)acrylaldehyde
(50 mg, 0.22 mmol) in THF (2 mL), methyl 2-(bromomethyl)acrylate
(43 mg, 0.24 mmol) and Zn--Cu couple powder (60 mesh, 25 mg) were
added. The reaction was stirred overnight at room temperature. At
the end, the mixture was filtrated in order to get rid of Zn--Cu
powder and the solvent was evaporated under vacuum. The crude
product was subjected to flash chromatography (Hex/EtOAc=50/50) to
give the desired compound in 94% yield.
[0228] Yield: 94%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.60
(m, 3H), 6.26 (t, J=2.7 Hz, 1H), 6.09 (dd, J=15.6, 6.9 Hz, 1H),
5.67 (t, J=2.4 Hz, 1H), 5.11 (q, J=6.9, 1H), 3.86 (s, 6H), 3.83 (s,
3H), 3.24 (ddt, J=17.1, 8.1, 2.4 Hz, 1H), 2.81 (m, 1H); .sup.13C
NMR (75 MHz, CDCl.sub.3) .delta. 170.0, 153.3, 138.4, 134.0, 133.1,
131.2, 126.1, 122.4, 103.8, 77.3, 60.8, 56.0, 34.2; IR (thin film,
cm.sup.-1) 2939, 1762, 1583, 1507, 1464, 1421, 1329, 1265, 1242,
1184, 1126, 1002, 964, 816, 730, 701, 624, 528, 423; m/z found:
291.31 [M+H.sup.+]; HRMS (FAB) calcd for C.sub.16H.sub.18O.sub.5:
290.1154; found 290.1154.
##STR00125##
[0229] Experimental Procedure:
[0230] To a solution of dihydropiperlongumine (346 mg, 1.08 mmol)
in THF (8 mL) was added LDA (1.5 equiv., 1.625 mmol, prepared
freshly from n-BuLi and i-Pr.sub.2NH) at -78.degree. C. After 30
min, methyl carbonochloride (113 mg, 1.19 mmol) was added dropwise.
The reaction mixture was slowly warmed up to room temperature and
stirred for overnight. The reaction was then quenched with
saturated aqueous NH.sub.4Cl solution (10 mL): the aqueous layer
was extracted with CH.sub.2Cl.sub.2 (3.times.10 mL) and the organic
layers were collected, washed with saturated aqueous NaCl solution
(30 mL) and dried over MgSO.sub.4. The solution was filtrated and
the solvent was evaporated under vacuum. The crude was subjected to
flash chromatography (hexane/ethyl acetate=3/1) to afford the
desired product in 49% yield.
[0231] Yield: 49%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.66
(d, J=15.6 Hz, 1H), 7.32 (d, J=15.3 Hz, 1H), 6.78 (s, 2H), 3.88 (s,
6H), 3.87 (s, 3H), 3.81 (s, 3H), 3.83 (m, 2H), 3.63 (t, J=6.9 Hz,
1H), 1.80-2.30 (m, 4H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
170.2, 170.0, 169.3, 153.4, 144.3, 130.5, 120.7, 105.7, 60.9, 56.2,
52.7, 51.4, 44.2, 24.4, 20.8; HRMS (FAB) calcd for
C.sub.19H.sub.23NO.sub.7: 377.1475; found 377.1475.
[0232] Experimental Procedure:
[0233] To a solution of the above carboxylate (76 mg, 0.20 mmol) in
THF (2 mL) was added NaH (6.1 mg, 0.24 mmol, 95%) at 0.degree. C.
After 30 min, PhSeCl (46.3 mg, 0.24 mmol) was added in one portion.
The reaction mixture was slowly warmed up to room temperature and
stirred for overnight. The reaction was then quenched with
saturated aqueous NH.sub.4Cl solution (5 mL): the aqueous layer was
extracted with CH.sub.2Cl.sub.2 (3.times.5 mL) and the organic
layers were collected, washed with saturated aqueous NaCl solution
(15 mL) and dried over MgSO.sub.4. The solution was filtrated and
the solvent was evaporated under vacuum. The crude was subjected to
flash chromatography (hexane/ethyl acetate=100/0 to 85/15) to
afford the desired product in 61% yield.
[0234] Yield: 61%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.66-7.70 (m, 3H), 7.42-7.46 (m, 1H), 7.32-7.37 (m, 2H), 7.25 (d,
J=15.6 Hz, 1H), 3.91 (s, 6H), 3.89 (s, 3H), 3.81 (s, 3H), 3.64-3.70
(m, 2H), 3.31-3.37 (m, 1H), 1.97-2.08 (m, 1H), 1.91-1.96 (m, 1H),
1.66-1.80 (m, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.3,
170.1, 169.1, 153.3, 144.4, 140.2, 138.4, 130.4, 129.9, 128.9,
126.2, 120.2, 105.7, 60.9, 56.9, 56.2, 53.4, 43.9, 32.0, 20.8; m/z
found: 533.97 [M+H.sup.+].
[0235] Experimental Procedure:
[0236] To a solution of the above senelide (60 mg, 0.11 mmol) in
CH.sub.2Cl.sub.2, (2 mL) was added H.sub.2O.sub.2 (0.029 mL, 0.28
mmol, 30%) at 0.degree. C. After 40 min, the reaction mixture was
slowly warmed up to room temperature and stirred for another 30
min. The reaction was then quenched with saturated aqueous
NH.sub.4Cl solution (5 mL): the aqueous layer was extracted with
CH.sub.2Cl.sub.2, (3.times.5 mL) and the organic layers were
collected, washed with saturated aqueous NaCl solution (15 mL) and
dried over MgSO.sub.4. The solution was filtrated and the solvent
was evaporated under vacuum. The crude was subjected to flash
chromatography (hexane/ethyl acetate=100/0 to 60/40) to afford the
desired product in 57% yield.
[0237] Yield: 57%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
7.66-7.71 (m, 2H), 7.50 (d, J=15.3 Hz, 1H), 6.79 (s, 2H), 4.05 (d,
J=6.3 Hz, 2H), 3.88 (s, 6H), 3.87 (s, 3H), 3.85 (s, 3H), 2.57-2.63
(m, 2H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 168.7, 164.1,
162.2, 153.3, 151.2, 144.8, 140.2, 130.5, 130.1, 120.7, 105.7,
60.9, 56.2, 52.5, 41.2, 24.9; HRMS (FAB) calcd for
C.sub.19H.sub.21NO.sub.7: 375.1318; found 375.1319.
##STR00126##
[0238] Experimental Procedure:
[0239] A mixture of piperlongumine (89 mg, 0.28 mmol) and methyl
2-mercaptoacetate (89 mg, 0.84 mmol) in DMSO (1 mL) was stirred at
room temperature for overnight. The reaction was then quenched with
saturated aqueous NH.sub.4Cl solution (5 mL): the aqueous layer was
extracted with CH.sub.2Cl.sub.2 (3.times.5 mL) and the organic
layers were collected, washed with saturated aqueous NaCl solution
(15 mL) and dried over MgSO.sub.4. The solution was filtrated and
the solvent was evaporated under vacuum. The crude was subjected to
flash chromatography (hexane/ethyl acetate=100/0 to 75/25) to
afford the desired product in 62% yield.
[0240] Yield: 62%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.62
(d, J=15.6 Hz, 1H), 7.32 (d, J=15.6 Hz, 1H), 6.76 (s, 2H),
3.99-4.10 (m, 1H), 3.86 (s, 6H), 3.85 (s, 3H), 3.73 (s, 3H),
3.62-3.68 (m, 1H), 3.33-3.39 (m, 1H), 3.29 (s, 2H), 3.00 (dd,
J=17.1, 5.4 Hz, 1H), 2.61 (dd, J=17.1, 9.0 Hz, 1H), 2.57-2.32 (m,
1H), 1.79-1.92 (m, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
171.0, 170.3, 169.0, 153.3, 144.0, 140.0, 130.4, 120.7, 105.5,
60.9, 56.1, 52.5, 42.4, 40.9, 37.4, 32.0, 28.8; HRMS (FAB) calcd
for C.sub.20H.sub.25NO.sub.7S: 423.1352; found 423.1351.
##STR00127##
[0241] Experimental Procedure:
[0242] A mixture of homo-piperlongumine (36 mg, 0.11 mmol) and
methyl 2-mercaptoacetate (35 mg, 0.22 mmol) in DMSO (2.5 mL) was
stirred at room temperature for 3 days. The reaction was then
quenched with saturated aqueous NH.sub.4Cl solution (5 mL): the
aqueous layer was extracted with CH.sub.2Cl.sub.2 (3.times.5 mL)
and the organic layers were collected, washed with saturated
aqueous NaCl solution (15 mL) and dried over MgSO.sub.4. The
solution was filtrated and the solvent was evaporated under vacuum.
The crude was subjected to flash chromatography (hexane/ethyl
acetate=80/20) to afford the desired product in 76% yield.
[0243] Yield: 76%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.62
(d, J=15.6 Hz, 1H), 7.29 (d, J=15.3 Hz, 1H), 6.78 (s, 2H),
3.94-4.01 (m, 2H), 3.88 (s, 6H), 3.86 (s, 3H), 3.74 (s, 3H),
3.25-3.42 (m, 3H), 3.07-3.09 (m, 2H), 2.08-2.19 (m, 1H), 1.86-2.01
(m, 2H), 1.69-1.80 (m, 1H); .sup.13C NMR (75 MHz, CDCl.sub.3)
.delta. 174.3, 170.5, 168.6, 153.3, 143.8, 140.0, 130.6, 121.0,
105.5, 60.9, 56.1, 52.5, 44.6, 43.3, 40.0, 34.9, 32.3, 26.2; m/z
found: 438.48; [M+H.sup.+]; HRMS (FAB) calcd for
C.sub.21H.sub.27NO.sub.7S: 437.1508; found 437.1507.
##STR00128##
[0244] Experimental Procedure:
[0245] A mixture of cyclopropylalkyne piperlongumine (93 mg, 0.244
mmol) and methyl 2-mercaptoacetate (155 mg, 1.46 mmol) in DMSO (3
mL) was stirred at room temperature for 3 days. The reaction was
then quenched with saturated aqueous NH.sub.4Cl solution (5 mL):
the aqueous layer was extracted with CH.sub.2Cl.sub.2 (3.times.5
mL) and the organic layers were collected, washed with saturated
aqueous NaCl solution (15 mL) and dried over MgSO.sub.4. The
solution was filtrated and the solvent was evaporated under vacuum.
The crude was subjected to flash chromatography (hexane/ethyl
acetate=80/20) to afford the desired product as an inseparable 2/1
mixture in 20% yield.
[0246] Yield: 15%; .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.71
(d, J=15.6 Hz, 1H, major), 7.67 (d, J=15.6 Hz, 1H, minor), 7.53 (d,
J=15.6 Hz, 1H, major), 7.47 (d, J=15.6 Hz, 1H, minor), 6.97 (t,
J=4.2 Hz, 1H, major), 6.93 (t, J=4.5 Hz, 1H, minor), 6.81 (s, 2H,
major), 6.80 (s, 2H, minor), 5.56 (d, J=10.2 Hz, 1H, major), 5.48
(d, J=9.9 Hz, 1H, minor), 4.10 (t, J=6.6 Hz, 2H, major), 4.04 (t,
J=6.6 Hz, 2H, minor), 3.90 (s, 6H, minor), 3.89 (s, 6H, major),
3.87 (s, 3H, major and minor), 3.70 (s, 3H, major), 3.69 (s, 3H,
minor), 3.33 (s, 2H, minor), 3.31 (s, 2H, major), 2.56-2.62 (m, 2H,
major), 2.48-2.54 (m, 2H, minor), 1.34-1.45 (m, 1H, major and
minor), 0.87-0.93 (m, 2H, minor), 0.76-0.81 (m, 2H, major),
0.49-0.54 (m, 2H, major and minor); .sup.13C NMR (75 MHz,
CDCl.sub.3) .delta. major 170.3, 169.1, 164.5, 153.3, 145.4, 144.9,
144.3, 140.1, 133.5, 130.6, 124.5, 121.1, 105.7, 60.9, 56.3, 52.3,
41.9, 35.6, 24.9, 12.9, 7.8; HRMS (FAB) calcd For
C.sub.25H.sub.29NO.sub.7S: 487.1665; found 487.1669.
##STR00129##
[0247] Experimental Procedure:
[0248] To the solution of 5,6-dihydropyridine-2(1H)one (64.1 mg,
0.66 mmol, 2.2 equiv) in THF (3.0 mL, 0.1 M) at -78.degree. C. was
added solution of n-BuLi in hexanes (413 .mu.L, 1.6 M, 0.66 mmol,
2.2 equiv) and stirred for 15 minutes. To this solution was added
1,4-phenylenediacryloyl chloride (77 mg, 0.30 mmol) and the
reaction was stirred at -78.degree. C. for 3 hours. Reaction
mixture was diluted with ethyl acetate, quenched with aqueous
ammonium chloride, extracted with EtOAc, washed with brine, dried
with anhydrous sodium sulfate and purified by column chromatography
using hexanes-ethyl acetate gradient (0 to 80% EtOAc), yielding
81.3 mg of the product (72%).
[0249] For (E)-N-methacryloylbut-2-enamide (BRD7991; Cpd. 76).
##STR00130##
[0250] .sup.1H NMR (300 MHz, CHLOROFORM-d) .delta. 8.35 (s, 1H),
7.25-6.90 (m, 2H), 5.79 (d, J=0.6 Hz, 1H), 5.56 (dd, J=3.0, 1.4 Hz,
1H), 1.94 (s, 3H), 1.89 (dd, J=6.5, 1.2 Hz, 3H); .sup.13C NMR (75
MHz, CHLOROFORM-d) .delta. 167.4, 166.8, 147.1, 140.1, 124.3,
122.7, 18.7, 18.6. Exact mass (M+Na).sup.+ calc'd: 176.0687, found
176.0690.
[0251] Experimental Procedure:
[0252] To a suspension of (E)-but-2-enamide (1.0 equiv) in 3.5 mL
THF was added n-BuLi in hexanes (2.5 M, 1.2 equiv) slowly at
-78.degree. C. The reaction mixture was warmed up to room
temperature and stirred for 8 h before a solution of methacryloyl
chloride (2.0 equiv) in 2 mL of THF was added drop wise at
-78.degree. C. The reaction mixture was allowed to warm up to rt
overnight. The crude reaction mixture was diluted with ethyl
acetate, and quenched with cold NH.sub.4Cl.sub.(aq, sat), and the
organic layer separated. The aqueous layer was extracted twice with
ethyl acetate. The combined organic layers were washed with brine
and dried over MgSO.sub.4. After filtration and evaporation under
reduced pressure, the residue was purified by column chromatography
to yield the pure compound.
Example 2
Methods
[0253] Cell culture.
[0254] HeLa, U2OS, EJ, and H1703 were obtained from ATCC. HeLa and
U2OS were cultured in DMEM+10% FBS in a 37.degree. C. incubator (5%
CO.sub.2); H1703 was cultured in RPMI+10% FBS, and EJ in McCoy's
5A+10% FBS. HEC108 were obtained from the Broad Institute/Novartis
Cancer Cell Line Encyclopedia and cultured in EMEM+15% FBS. BJ
(human fibroblasts) were obtained from ATCC, while BJ-ELR (a fully
transformed derivative containing hTERT, large-T antigen, and
activated H-RAS) were a gift of Prof. Brent Stockwell, Columbia
University, New York, N.Y. Both lines were maintained in 4:1
DMEM:M1999 supplemented with 15% FBS. BJ were maintained below 70%
confluency and used below passage 8.
[0255] ROS Assay.
[0256] Cells were plated at 5,000 cells/well in black 384-well
plates (Corning) and allowed to attach overnight. The next day (ca.
90% confluency), dilutions of compounds in DMSO were added by pin
transfer (CyBio Vario, 100 mL per well), and incubated for 90
minutes. Media was changed using a Thermo Multidrop Combi liquid
handler to phenol red-free DMEM containing 10 .mu.M
CM-H.sub.2DCF-DA and 10 .mu.g/mL Hoechst 33342. Following
incubation for 15-30 minutes, cells were washed twice with PBS.
Images were obtained using an IX_Micro automated fluorescence
microscope (Molecular Devices). Quantitation of pixel intensity was
performed using MetaXpress software and signal intensity was
calculated relative to wells in the same plate treated with
DMSO.
[0257] ATP Assay.
[0258] Cells were plated at 1,000 per well in white 384-well plates
and allowed to attach overnight. After addition of compounds by pin
transfer, plates were incubated 48 h. At that time, media was
removed and replaced with a solution of CellTiter-Glo reagent
(Promega) in PBS. After ten minutes, luminescence was read using an
EnVision multilabel plate reader (Perkin-Elmer) and signal
intensity was calculated relative to in-plate DMSO control
wells.
[0259] Glutathione Assay.
[0260] Cells were plated at 1,000 per well in white 384-well plates
and allowed to attach overnight. After addition of compounds by pin
transfer, plates were incubated for 3 h (EJ) or 6 h (HeLa). Cells
were washed with PBS, and total glutathione was measured using
GSH/GSSG-Glo, according to manufacturer's instructions (Promega).
Luminescence was measured with an EnVision multilabel plate
reader.
[0261] Immunofluorescence Detection of Glutathionylated
Proteins.
[0262] Cells were plated at 3,000 (HeLa) or 5,000 (EJ) per well and
allowed to attach overnight. After addition of compounds by pin
transfer, plates were incubated for between 10 minutes and 6 h. At
the appropriate time, cells were fixed with 1% paraformaldehyde in
PBS (20 minutes), permeabilized 30 minutes withPBS+0.1% TritonX-100
("PBST"), and blocked 30 minutes with PBST+2% BSA. Primaryantibody
(Ms anti-glutathione, Abcam Ab19534) was added (1:1250 in PBST+2%
BSA) and incubated at 4.degree. C. overnight. Following two washes
with PBST, cells were incubated at RT 1 h in the dark with
secondary antibody solution (Cy-2 or Cy-3-conjugated goat
anti-mouse, Jackson Immuno chemicals, 1:500, plus Hoechst 33342, 10
.mu.g/ml in PBST+2% BSA). Following two washes with PBST, images
were collected using an IX_Micro automated fluorescencemicroscope
(Molecular Devices). Quantitation of pixel intensity was performed
as above.
[0263] Assessment of Cancer/Normal Selective Toxicity.
[0264] BJ vs. BJ-ELR: In 12-well dishes, BJ (33,000 cells/well) or
BJ-ELR (25,000 cells/well) were seeded and allowed to grow to
40-50% confluency (24-36 h). Compound solutions in DMSO were added
(0.2% DMSO final) and incubated 48 h. Cells were fixed with 2%
paraformaldehyde (15 minutes), followed by staining with 0.01%
aqueous crystal violet (30 minutes). Cells were washed twice with
water and allowed to dry overnight. Once dry, the stain was
resolubilized using ethylene glycol (2 mL per well, 2-16 hr with
shaking). When cells retained no stain, duplicate samples of 50
.mu.L were then transferred to wells of a 384-well plate.
Absorbance at 540 nm was measured with an EnVision plate reader,
and relative viability was calculated relative to DMSO-treated
control wells.
Example 3
Chemical Reactivity of Piperlongumine and its Analogs
[0265] As PL contains multiple potentially electrophilic sites that
may influence its actions on cells, we assessed the chemical
reactivity of PL and several of our analogs using methyl
thioglycolate as a representative achiral thiol nucleophile.
Treatment of PL with 3 equivalents of methyl thioglycolate in DMSO
provided the product of conjugate addition at C3 in 62% yield (FIG.
1C). No addition was observed at C8 by LC-MS or .sup.1H NMR, and
2,3-dihydropiperlongumine (Table 1, entry 2) was unreactive under
these conditions. We next assessed whether PL analogs with
modifications proximal to the highly reactive C2-C3 olefin yield
similar patterns of reactivity. Substitution of methyl at C2 (Table
1, entry 3) ablated hetero-conjugate addition, indicating that C2
alkyl substituents can impede reaction at C3. A ring-expanded
cycloheptenimide analog (PL-7; Table 1, entry 4) also provided the
expected hetero-conjugate addition product at C3 (74% yield), while
C2 alkyne 4 (PL-cPr; Table 1, entry 5) underwent further reaction
to provide a rearranged thiol enol ether product (FIG. 9).
TABLE-US-00001 TABLE 1 Effects on cellular ATP levels for PL and
selected PL analogs in two cell lines. EC.sup.50 EC.sup.50 H1703
HeLa (.mu.M) (.mu.M) 1 ##STR00131## PL 2.8 7.1 2 ##STR00132##
PL-2,3H.sub.2 n.t. n.t. 3 ##STR00133## n.t. n.t. 4 ##STR00134##
PL-7 1.9 5.1 5 ##STR00135## PL-cPr 0.7 1.3 6 ##STR00136##
PL-H.sub.2 17.5 n.t. 7 ##STR00137## PL-MTG 7.9 >20 8
##STR00138## n.t. n.t. 9 ##STR00139## 14 >20 10 ##STR00140## 18
>20 11 ##STR00141## PL-DHN 14 n.t. 12 ##STR00142## 3.5 7.1 13
##STR00143## 4.0 7.5 14 ##STR00144## 3.6 6.8 15 ##STR00145## 6.5 15
16 ##STR00146## PL-SO.sub.2 2.7 4.1 17 ##STR00147## PL-FPh 0.4 1.0
18 ##STR00148## n.t. n.t.
[0266] Effects on cellular ATP levels for selected PL analogs in
two cell lines (H1073 and HeLa). n.t. indicates no loss of
viability at 20 .mu.M; >20 .mu.M indicates less than 50%
decrease in viability observed at 20 .mu.M. Column 1: Entry number;
Column 3: EC.sub.50 H1703 (.mu.M); Column 4: EC.sub.50 HeLa
(.mu.M).
Example 4
Cellular Actions
[0267] We next sought to determine the performance of the synthetic
analogs relative to PL in cells. Changes in ROS levels were
assessed 90 minutes after compound treatment by automated
microscopy using the redox-sensitive dye CM-H.sub.2DCF-DA, which
shows greatest sensitivity to hydroxyl radical and other highly
reactive species. As a second assay, effects of compound treatments
on ATP levels, a surrogate for cell viability, were measured after
48 hours. (A second assay monitoring cellular reducing equivalents
as a surrogate for viability gave closely correlated results. See
Example 9.) Both assays were performed in 384-well plates on two
human cancer cell lines, H1703 (lung) and HeLa (cervix).
[0268] As PL contains multiple electrophilic sites and can undergo
hetero-conjugate addition with small-molecule thiols, we
hypothesized that the electrophilicity of PL might be central to
its bioactivity. Our first analogs sequentially eliminated the two
reactive .alpha.,.beta.-unsaturated olefins (Table 1, entries 1-2,
6. Table 1 includes viability data for representative analogs in
two cell lines). For viability and ROS dose-response data for all
80 analogs, see Example 9. 2,3-dihydropiperlongumine (PL-2,3H2,
Table 1, entry 2), lacking the C2-C3 olefin, neither elevated ROS
levels nor decreased viability of the two cell lines tested,
demonstrating that this functionality is essential for PL's
biological activity (FIG. 2). By contrast, removal of the C7-C8
olefin (PL-H2) led to substantial reductions in toxicity (FIG. 2A),
but did not diminish ROS elevation (Table 1, entry 6; FIG. 2B, C).
These results indicate that while the presence of the C2-C3 olefin
is sufficient to elevate ROS, both olefins are necessary to
recapitulate the level of cellular toxicity observed for PL.
[0269] The study of additional analogs confirmed that modifications
diminishing the reactivity of the C2-C3 olefin yielded compounds
with minimal activity in these assays (Table 1, entries 7, 8).
Piperlongumine-thiol adduct PL-MTG, a potential PL pro-drug, was
found to be substantially less potent than PL at inducing cancer
cell death and elevating ROS (Table 1, entry 7).
[0270] Additional analogs disrupting the electrophilicity of the
C7-C8 olefin by steric blockade or cyclization to an aromatic
heterocycle showed substantially diminished toxicity in cells
(Table 1, entries 9-11). Together with 7,8-dihydropiperlongumine
PL-H2 discussed above, these analogs highlight the need for both
Michael acceptors to observe potent cell death.
[0271] Although the C2-C3 and C7-C8 olefins appear critical for
PL's actions on cells, many modifications can be made at positions
distal from these olefins without greatly affecting performance in
our ROS and ATP assays. Modification of the aromatic substituents
of PL was largely without effect: all three aryl methoxyl groups
could be removed and replaced with a variety of substituents at the
ortho, meta, or para positions without substantially altering
elevation of ROS or cellular toxicity (Table 1, entries 12, 13).
Likewise, substitution at C5 or C4 with aromatic, heteroaromatic,
and some alkyl substituents provided analogs of comparable potency
to PL (Table 1, entries 14, 15). Ring expansion of the
dihydropyridone, replacement of the C6 carbonyl with a sulfonyl
moiety, and several other modifications also were largely neutral
(Table 1, entry 4, 16).
[0272] Analogs with substituents at C2 provided a wide range of
activities. A variety of alkynyl substituents at C2 induced cell
death more potently than PL, with EC50 values as low as 0.4.mu.M
(Table 1, entries 5, 17). However, other substituents at C2 were
uniformly less potent than PL, with C2 alkyl or aryl groups lacking
activity in both assays (Table 1, entries 3, 18). The diminished
chemical reactivity of analogs with C2 alkyl substituents may be
one factor contributing to the observed lack of cellular activity
(FIG. 9).
[0273] Since both the C2-C3 and C7-C8 olefins are necessary to
observe the levels of toxicity seen for PL, we speculated that
multivalency--the ability to interact with multiple cellular
targets or a single cellular target at more than one
location--might alter toxicity in this system (13-16). Thus, we
synthesized a structurally analogous PL `monomer`, `dimer`, and
`trimer` using a Mitsunobu inversion approach (FIG. 3A, S1).
Remarkably, the PL dimer (PL-DI) showed roughly 10-fold greater
potency in the ATP and ROS assays relative to a closely related
monomeric analog (PL-MON) (FIG. 3B, C). Moreover, a PL trimer
(PL-TRI) was found to be two-fold more potent than the dimeric
analog.
Example 5
Separation of ROS Elevation from Cellular Toxicity
[0274] For PL and many analogs, doses at which ROS and cellular
toxicity are elevated are closely correlated. However, two series
of analogs appear to decouple the elevation of ROS and cell death.
In both cell lines tested, 7,8-dihydropiperlongumine (PL-H2) and a
dihydronaphthalene analog (PL-DHN) led to robust enhancement of ROS
levels but to diminished cell death relative to PL (FIGS. 2, 4).
Conversely, various analogs bearing alkynes at C2 showed greatly
enhanced toxicity (FIG. 4A) without altering potency for the
elevation of ROS (FIG. 4B). Although this enhanced cell death could
be explained by C2-alkynyl analogs having additional toxic
mechanisms of action, we were surprised to find analogs like PL-H2
and PL-DHN that showed elevation of ROS comparable to PL but
greatly diminished cellular toxicity. A similar pattern was
observed in two additional cancer cell lines (U2OS, osteosarcoma;
HEC108, endometrial), indicating that the observed decoupling of
ROS and cell death may be general (FIG. S2). Although elevation of
cellular ROS likely places cancer cell lines under enhanced
oxidative stress, this stress appears insufficient in some cases to
induce cell death.
Example 6
Additional Cellular Actions
[0275] Beyond elevation of ROS, PL affects other cellular markers
of oxidative stress, including depletion of glutathione (5). We
next characterized a subset of analogs in additional oxidative
stress assays to identify phenotypes that might correlate with
cellular toxicity more closely than ROS elevation. Using a
luminescence-based assay for cellular glutathione (GSH/GSSG-Glo) in
the EJ bladder carcinoma line (5), a similar decrease in total
cellular glutathione (ca. 60%) was observed for PL and two analogs
with diminished toxicity (PLH2, PL-DHN; FIG. 5A). Decreases in
total glutathione were also observed for all three compounds in
HeLa cells, with PL most effective (FIG. S4). We also examined an
additional oxidative stress phenotype, protein glutathionylation,
using an immunofluorescence approach that relies on a monoclonal
antibody recognizing glutathione. In HeLa cells, we observed large
and rapid elevations in protein glutathionylation for PL and its
potently toxic cyclopropyl alkynyl analog PL-cPr (FIG. 5B, C, FIG.
12). However, no elevation in protein glutathionylation was
observed for PL-H2, and minimal elevation was observed for PL-DHN.
Examination of our 80-analog set in both HeLa and EJ cells suggests
that an unhindered, chemically reactive C7-C8 olefin is necessary
for elevation of protein glutathionylation. Analogs with an
unreactive or absent C7-C8 olefin, modifications that also diminish
toxicity, show minimal elevation of protein glutathionylation (see
Example 9). Similarly, several small molecules unrelated to
piperlongumine bearing two Michael acceptor functionalities
elevated glutathionylation, while nine other small molecules with a
single electrophilic site did not (FIG. S6). Elevation of protein
glutathionylation also correlated with toxicity, as toxic PL
analogs bearing multiple Michael acceptor functionalities showed
robust protein glutathionylation.
[0276] Additionally, although proteins are commonly
glutathionylated during periods of oxidative stress via readily
reversible disulfide bond linkages (17, 18), the protein
gluathionylation observed following PL treatment could not be
reversed by treatment with 0.1 M dithiothreitol, indicating a role
or roles for non-disulfide covalent attachments. By contrast, the
elevation of protein glutathionylation observed following treatment
with glutathione disulfide (GSSG) was strikingly reversed by
treatment with 0.1 M dithiothreitol (Supporting FIG. 5).
Example 7
Selectivity for Cancer Cells Over Nontransformed Cells
[0277] We evaluated whether our analogs, like PL, could selectively
target cancer cells over nontransformed cells using an established
isogenic model of tumorigenesis (5). Such models rely on serial
transduction of primary human cell types with defined genetic
factors to create an engineered cancer cell line. We compared BJ
human fibroblasts with the BJ-ELR line, which is fully transformed
by the addition of hTERT, large-T antigen, and an oncogenic
HRAS-V12 allele (19). Eight representative analogs were evaluated
by crystal violet staining for cell numbers.
Phenethylisothiocyanate and parthenolide, two electrophilic small
molecules previously shown to be selectively toxic to cancer cells
by a mechanism involving ROS elevation (8, 12), also showed
selectivity in this assay. Initial screening of the eight analogs
established that most retained a degree of selective toxicity in
this isogenic cell line pair, although some were inferior to PL
(FIG. 13). Further testing confirmed that four analogs showed
selectivity comparable to PL (PL-7, PL-DI, PL-TRI, and sulfonimide
derivative PL-SO2), with the ring-expanded analog PL-7 being the
most selective (FIG. 6, 14, 18).
[0278] Similar experiments also indicated that certain iPLA analogs
could exhibit improved selectivity towards certain transformed
cells or cancer cells relative to non-transformed and non-cancer
cells, respectively (see data for "Sul", "7 ring", "Dimer", and
"Trimer" in FIGS. 18, and 19).
Example 8
Summary and References
[0279] By synthesizing and testing an array of PL analogs, we have
identified the C2-C3 olefin as a key pharmacophore, with the C7-C8
olefin also playing a significant role in determining toxicity
(FIG. 7A). Although a wide range of modifications at positions
distal to these olefins is largely neutral, modifications expected
to impair the reactivity of these olefins diminish analogs' effects
on cells. The C2-C3 olefin reacts with a small-molecule thiol under
neutral conditions in DMSO, but exposure of the resulting PL-thiol
adduct to cells was largely without effect. Addition of a C2 methyl
group ablates both chemical reactivity in vitro and all observed
cellular phenotypes, further supporting the necessity of reactivity
at C3 for actions in cells. Although addition of thiols at C8 was
not observed using our neutral in vitro conditions, the presence of
cellular thiolate nucleophiles or the enhanced effective molarity
following addition of a cellular nucleophile at C3 may greatly
enhance the rate of thiol addition at C8.
[0280] As both olefins appeared necessary for PL's toxicity to
cells, we evaluated several oligomers of PL to explore further the
role of multivalent electrophilicity in determining toxicity.
Notably, a PL dimer resulted in a nearly ten-fold increase in
toxicity. The ability to cross-link additional cellular
nucleophiles may limit the reversibility of compound binding or
cause more extensive disruption of the structure and function of
targeted proteins relative to PL itself. A PL trimer was only
two-fold more potent than the analogous dimer, suggesting
diminishing returns for addition of further PL units. These highly
potent oligomers, as well as several other analogs, also retained
similar selectivity for transformed cells as observed for PL in an
isogenic model of tumorigenesis.
[0281] As noted above, PL analogs with only a single electrophilic
moiety showed diminished cell death but often gave rise to
substantial increases in ROS, suggesting that ROS elevation may not
be sufficient or even necessary for cell death in some cellular
contexts. Although the specific ROS measured and their subcellular
localization may vary between analogs, PL and its active analogs
are likely capable of reaction with a variety of cellular protein
thiols, including some that may contribute to cell death
independent of elevation of ROS levels.
[0282] We also assessed the performance of our analogs in
additional oxidative stress assays. Several analogs with varying
degrees of toxicity showed a similar ability to deplete cellular
glutathione. As previous reports have established that substantial
reductions in glutathione (for example, as induced by the
glutathione biosynthesis inhibitor BSO) need not result in cell
death, the contribution of glutathione depletion to PL's cellular
toxicity remains unclear (20-22). Additionally, we note that
reported estimates of HeLa cell volume (2,600 .mu.m.sup.3) (23) and
typical concentrations of cellular reduced glutathione (ca. 5 mM)
(24) suggest that PL is present at quantities greatly in excess of
cellular glutathione under our assay conditions (1,000 cells per
well, 50 .mu.l per well). As such, direct conjugation of PL with
glutathione at C3 is a plausible explanation for the observed
decrease in total glutathione.
[0283] Treatment with PL or analogs with two reactive electrophilic
sites also gives rise to enhanced glutathionylation of cellular
proteins, while analogs with a single Michael acceptor did not. We
propose that formation of a covalent complex linking glutathione,
PL, and a glutathione binding protein via both PL's electrophilic
Michael acceptor functionalities can account for these observations
(FIG. 7B). Without seeking to be limited by theory, we imagine a
sequence involving first a Michael addition of glutathione to PL's
more electrophilic C(2)-C(3) olefin followed by the formation of a
noncovalent complex between the PL-glutathione adduct and a
glutathione-binding protein, and finally a Michael addition of a
nucleophilic residue of the glutathione-binding protein to the less
electrophilic C(7)-C(8) olefin that is accelerated by the formation
of the complex. Mechanistically analogous protein glutathionylation
under conditions of electrophilic stress has been observed recently
for the metabolic byproduct 4-oxo-nonenal (25) and the
chemotherapeutic busulfan (26), both of which are bivalent
electrophiles. Such a model also provides a chemical rationale for
the results of an unbiased quantitative proteomics analysis of
proteins binding to PL, which identified numerous
glutathione-binding proteins among the highest confidence
interactions (5). We note that there exist other naturally
occurring, biologically active small molecules with two sites of
reactivity--one with greater and the other with lesser
electrophilicity. Thus, it is possible that the proposed mechanism,
where a small organic molecule is inserted between glutathione and
a protein in the protein glutathionylation process, has generality
beyond PL. Although not measured in this study, PL may also
covalently modify additional cellular proteins. Notably, proteins
that do not bind glutathione were also high-signal outliers in the
reported quantitative proteomic analysis, including four proteins
previously shown to be modified following treatment with
electrophiles (PRDX1; RPS5; VIM; AHNAK) (5, 27-30). Taken together,
our observations suggest that elevation of protein
glutathionylation or other cellular cross-linking events may be a
feature of cells treated with PL more closely associated with
cellular toxicity than elevation of ROS or glutathione depletion.
Further proteomic analyses will be required to identify specific
protein glutathionylation events and proteins that interact with
analogs of markedly enhanced (PL-DI, PL-TRI) or diminished (PL-H2)
electrophilicity and toxicity (31). These studies establish a
central role for multivalent electrophilicity in the chemical
biology of PL and related compounds, and indicate that both
electrophilic and oxidative stress phenotypes can contribute to
PL's promising cancer-selective toxicity.
REFERENCES
[0284] 1. Lee A C, et al. (1999) Ras proteins induce senescence by
altering the intracellular levels of reactive oxygen species. J
Biol Chem 274(12):7936-7940. [0285] 2. Szatrowski T P & Nathan
C F (1991) Production of large amounts of hydrogen peroxide by
human tumor cells. Cancer Res 51(3):794-798. [0286] 3. Luo J,
Solimini N L, & Elledge S J (2009) Principles of cancer
therapy: oncogene and non-oncogene addiction. Cell 136(5):823-837.
[0287] 4. Fruehauf J P & Meyskens F L, Jr. (2007) Reactive
oxygen species: a breath of life or death? Clin Cancer Res
13(3):789-794. [0288] 5. Raj L, et al. (2011) Selective killing of
cancer cells by a small molecule targeting the stress response to
ROS. Nature 475(7355):231-234. [0289] 6. Wondrak G T (2009)
Redox-directed cancer therpeutics: molecular mechanisms and
opportunities. Antioxid Redox Signal 11(12):3013-3069. [0290] 7.
Trachootham D, Alexandre J, & Huang P (2009) Targeting cancer
cells by ROSmediated mechanisms: a radical therapeutic approach?
Nat Rev Drug Discov 8(7):579-591. [0291] 8. Trachootham D, et al.
(2006) Selective killing of oncogenically transformed cells through
a ROS-mediated mechanism by beta-phenylethyl isothiocyanate. Cancer
Cell 10(3):241-252. [0292] 9. Huang P, Feng L, Oldham E A, Keating
M J, & Plunkett W (2000) Superoxide dismutase as a target for
the selective killing of cancer cells. Nature 407(6802):390-395.
[0293] 10. Shaw A T, et al. (2011) Selective killing of K-ras
mutant cancer cells by small molecule inducers of oxidative stress.
Proc Natl Acad Sci USA 108(21):8773-8778. [0294] 11. Dolma S,
Lessnick S L, Hahn W C, & Stockwell B R (2003) Identification
of genotypeselective antitumor agents using synthetic lethal
chemical screening in engineered human tumor cells. Cancer Cell
3(3):285-296. [0295] 12. Guzman M L, et al. (2007) An orally
bioavailable parthenolide analog selectively eradicates acute
myelogenous leukemia stem and progenitor cells. Blood
110(13):4427-4435. [0296] 13. Corson T W, Aberle N, & Crews C M
(2008) Design and Applications of Bifunctional Small Molecules Why
Two Heads Are Better Than One. ACS Chem Biol 3(11):677-692. [0297]
14. Dinkova-Kostova A T, et al. (2005) Extremely potent
triterpenoid inducers of the phase 2 response: correlations of
protection against oxidant and inflammatory stress. Proc Natl Acad
Sci USA 102(12):4584-4589. [0298] 15. Wissner A, et al. (2003)
Synthesis and structure-activity relationships of 6,7-disubstituted
4-anilinoquinoline-3-carbonitriles. The design of an orally active,
irreversible inhibitor of the tyrosine kinase activity of the
epidermal growth factor receptor (EGFR) and the human epidermal
growth factor receptor-2 (HER-2). J Med Chem 46(1):49-63. [0299]
16. West J D, Stamm C E, Brown H A, Justice S L, & Morano K A
(2011) Enhanced toxicity of the protein cross-linkers divinyl
sulfone and diethyl acetylenedicarboxylate in comparison to related
monofunctional electrophiles. Chem Res Toxicol 24(9):1457-1459.
[0300] 17. Dalle-Donne I, et al. (2008) Molecular mechanisms and
potential clinical significance of S-glutathionylation. Antioxid
Redox Signal 10(3):445-473. [0301] 18. Klatt P & Lamas S (2000)
Regulation of protein function by S-glutathiolation in response to
oxidative and nitrosative stress. Eur J Biochem 267(16):4928-4944.
[0302] 19. Hahn W C, et al. (1999) Creation of human tumour cells
with defined genetic elements. Nature 400(6743):464-468. [0303] 20.
Marengo B, et al. (2008) GSH loss per se does not affect
neuroblastoma survival and is not genotoxic. Int J Oncol
32(1):121-127. [0304] 21. Goto H, Yanagimachi M, Kajiwara R, Kuroki
F, & Yokota S (2007) Lack of mitochondrial depolarization by
oxidative stress is associated with resistance to buthionine
sulfoximine in acute lymphoblastic leukemia cells. Leuk Res
31(9):1293-1301. [0305] 22. Green R M, Graham M, O'Donovan M R,
Chipman J K, & Hodges N J (2006) Subcellular
compartmentalization of glutathione: correlations with parameters
of oxidative stress related to genotoxicity. Mutagenesis
21(6):383-390. [0306] 23. Zhao L, et al. (2008) Intracellular
water-specific MR of microbead-adherent cells: the HeLa cell
intracellular water exchange lifetime. NMR Biomed 21(2):159-164.
[0307] 24. Chakravarthi S, Jessop C E, & Bulleid N J (2006) The
role of glutathione in disulphide bond formation and
endoplasmic-reticulum-generated oxidative stress. EMBO Rep
7(3):271-275. [0308] 25. Zhu X, Gallogly M M, Mieyal J J, Anderson
V E, & Sayre L M (2009) Covalent crosslinking of glutathione
and carnosine to proteins by 4-oxo-2-nonenal. Chem Res Toxicol
22(6):1050-1059. [0309] 26. Cooper A J, Pinto J T, & Callery P
S (2011) Reversible and irreversible protein glutathionylation:
biological and clinical aspects. Expert Opin Drug Metab Toxicol
7(7):891-910. [0310] 27. Rabilloud T, et al. (2002) Proteomics
analysis of cellular response to oxidative stress. Evidence for in
vivo overoxidation of peroxiredoxins at their active site. J Biol
Chem 277(22):19396-19401. [0311] 28. Shenton D & Grant C M
(2003) Protein S-thiolation targets glycolysis and protein
synthesis in response to oxidative stress in the yeast
Saccharomyces cerevisiae. Biochem J 374(Pt 2):513-519. [0312] 29.
Huang B, Liao CL, Lin Y P, Chen S C, & Wang D L (2009)
S-nitrosoproteome in endothelial cells revealed by a modified
biotin switch approach coupled with Western blot-based
two-dimensional gel electrophoresis. J Proteome Res
8(10):4835-4843. [0313] 30. Koshiyama A & Imai K (2010)
Synthesis and evaluation of a fluorogenic reagent for proteomic
studies:
7-fluoro-N-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-4-sulfonamide
(DAABD-F). Analyst 135(8):2119-2124. [0314] 31. Liebler D C (2008)
Protein damage by reactive electrophiles: targets and consequences.
Chem Res Toxicol 21(1):117-128.
Example 10
EC50 Values for Cell Viability for Select Analogs
[0315] The effects of compound treatments on ATP levels, a
surrogate for cell viability, were measured after 48 hours
essentially as described in Example 2. Assays were performed in
384-well plates on two human cancer cell lines, H1703 (lung) and
HeLa (cervix).
TABLE-US-00002 TABLE 2 Effects on cellular ATP levels for PL and
selected PL analogs in two cell lines. EC.sub.50 EC.sub.50 H1703
HeLa No. Structure (.mu.M) (.mu.M) 1 ##STR00149## PL 2.8 7.1 2
##STR00150## PL-2,3H.sub.2 n.t. n.t. 3 ##STR00151## n.t. n.t. 4
##STR00152## PL-7 1.9 5.1 5 ##STR00153## PL-cPr 0.7 1.3 6
##STR00154## PL-H.sub.2 17.5 n.t. 7 ##STR00155## 2 2 8 ##STR00156##
3 6 9 ##STR00157## 4 7 10 ##STR00158## 2 4 11 ##STR00159## 6 7 12
##STR00160## 6 6 13 ##STR00161## n.t n.t 14 ##STR00162## 5 8 15
##STR00163## 10 20 16 ##STR00164## n.t n.t 17 ##STR00165## 1 3 18
##STR00166## 5 6 19 ##STR00167## n.t n.t. 20 ##STR00168## >20
n.t. 21 ##STR00169## n.t. n.t. 22 ##STR00170## 9 20 23 ##STR00171##
0.8 3 24 ##STR00172## >20 n.t. 25 ##STR00173## 7.9 >20 26
##STR00174## n.t. n.t. 27 ##STR00175## 14 >20 28 ##STR00176## 18
>20 29 ##STR00177## PL-DHN 14 n.t. 30 ##STR00178## 3.5 7.1 31
##STR00179## 0.9 2 32 ##STR00180## 1.5 4 33 ##STR00181## n.t n.t 34
##STR00182## 5 15 35 ##STR00183## 0.6 1.5 36 ##STR00184## 0.5 1.5
37 ##STR00185## 4 18 38 ##STR00186## 3 8 39 ##STR00187## 5 18 40
##STR00188## 1.5 5 41 ##STR00189## 0.1 0.3 42 ##STR00190## 12
>20 43 ##STR00191## 0.7 1.2 44 ##STR00192## n.t. n.t. 45
##STR00193## 3 5 46 ##STR00194## 13 >20 47 ##STR00195## n.t.
n.t. 48 ##STR00196## 0.7 3 49 ##STR00197## 4.0 7.5 50 ##STR00198##
3.6 6.8 51 ##STR00199## 6.5 15 52 ##STR00200## PL-SO.sub.2 2.7 4.1
53 ##STR00201## PL-FPh 0.4 1.0 54 ##STR00202## n.t. n.t. 55
##STR00203## 3 >20 56 ##STR00204## n.t n.t 57 ##STR00205## 4 9
58 ##STR00206## 8 11 59 ##STR00207## 1.5 3 60 ##STR00208## 2 4 61
##STR00209## 0.5 1.5 62 ##STR00210## 5 7 63 ##STR00211## 2 10 64
##STR00212## 7 10 65 ##STR00213## n.t n.t. 66 ##STR00214## n.t n.t.
67 ##STR00215## 0.2 0.5 68 ##STR00216## 0.4 0.9 69 ##STR00217## 1 2
70 ##STR00218## 10 >20 71 ##STR00219## n.t. n.t. 72 ##STR00220##
2.2 8.1 73 ##STR00221## 4.7 19 74 ##STR00222## 2.3 11 75
##STR00223## 0.91 4.1 76 ##STR00224## 8.0 >20 77 ##STR00225##
5.5 15 78 ##STR00226## 0.30 0.78 Effects on cellular ATP levels for
selected PL analogs in two cell lines (H1073 and HeLa). n.t.
indicates no loss of viability at 20 .mu.M; >20 .mu.M indicates
less than 50% decrease in viability observed at 20 .mu.M.
* * * * *